39-desmethoxy derivatives of rapamycin

ABSTRACT

The present invention relates to novel 39-desmethoxyrapamycin derivatives, methods for their production, and uses thereof. In a further aspect the present invention provides for the use of these 39-desmethoxyrapamycin derivatives in the treatment of cancer and/or B-cell malignancies, the induction or maintenance of immunosuppression, the treatment of transplantation rejection, graft vs. host disease, autoimmune disorders, diseases of inflammation, vascular disease and fibrotic diseases, the stimulation of neuronal regeneration or the treatment of fungal infections.

This application is a §371 application of PCT/GB2006/000853 filed Mar.10, 2006, which in turn claims priority to GB Application 0504994.5filed Mar. 11, 2005. The entire disclosure of each of the foregoingapplications is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to novel 39-desmethoxyrapamycinderivatives, methods for their production, and uses thereof. In afurther aspect the present invention provides for the use of these39-desmethoxyrapamycin derivatives in the treatment of cancer and/orB-cell malignancies, the induction or maintenance of immunosuppression,the treatment of transplantation rejection, graft vs. host disease,autoimmune disorders, diseases of inflammation, vascular disease andfibrotic diseases, the stimulation of neuronal regeneration or thetreatment of fungal infections.

BACKGROUND OF THE INVENTION

Rapamycin (sirolimus) (FIG. 1) is a lipophilic macrolide produced byStreptomyces hygroscopicus NRRL 5491 (Sehgal et al., 1975; Vézina etal., 1975; U.S. Pat. No. 3,929,992; U.S. Pat. No. 3,993,749) with a1,2,3-tricarbonyl moiety linked to a pipecolic acid lactone (Paiva etal., 1991). For the purpose of this invention rapamycin is described bythe numbering convention of McAlpine et al. (1991) in preference to thenumbering conventions of Findlay et al. (1980) or Chemical Abstracts(11^(th) Cumulative Index, 1982-1986 p60719CS).

Rapamycin has significant pharmacological value due to the wide spectrumof activities exhibited by the compound. Rapamycin shows moderateantifungal activity, mainly against Candida species but also againstfilamentous fungi (Baker et al., 1978; Sehgal et al., 1975; Vézina etal., 1975; U.S. Pat. No. 3,929,992; U.S. Pat. No. 3,993,749). Rapamycininhibits cell proliferation by targeting signal transduction pathways ina variety of cell types, e.g. by inhibiting signalling pathways thatallow progression from the G₁ to the S-phase of the cell cycle (Kuo etal., 1992). In T cells rapamycin inhibits signalling from the IL-2receptor and subsequent autoproliferation of the T cells resulting inimmunosuppression. The inhibitory effects of rapamycin are not limitedto T cells, since rapamycin inhibits the proliferation of many mammaliancell types (Brunn et al., 1996). Rapamycin is, therefore, a potentimmunosuppressant with established or predicted therapeutic applicationsin the prevention of organ allograft rejection and in the treatment ofautoimmune diseases (Kahan et al., 1991).40-O-(2-hydroxy)ethyl-rapamycin (SDZ RAD, RAD 001, Certican, everolimus)is a semi-synthetic analogue of rapamycin that shows immunosuppressivepharmacological effects and is also under investigation as an anticanceragent (Sedrani, R. et al., 1998; Kirchner et al., 2000; U.S. Pat. No.5,665,772, Boulay et al, 2004). Approval for this drug as animmunosuppressant was obtained for Europe in 2003. The rapamycin esterderivative CCI-779 (Wyeth-Ayerst) inhibits cell growth in vitro andinhibits tumour growth in vivo (Yu et al., 2001). CCI-779 is currentlyin Phase III clinical trials as a potential anti-cancer agent. The valueof rapamycin in the treatment of chronic plaque psoriasis (Kirby andGriffiths, 2001), the potential use of effects such as the stimulationof neurite outgrowth in PC12 cells (Lyons et al., 1994), the block ofthe proliferative responses to cytokines by vascular and smooth musclecells after mechanical injury (Gregory et al., 1993) and its role inprevention of allograft fibrosis (Waller and Nicholson, 2001) are areasof intense research (Kahan and Camardo, 2001). Recent reports revealthat rapamycin is associated with a lower incidence of cancer in organallograft patients on long-term immunosuppressive therapy than those onother immunosuppressive regimes, and that this reduced cancer incidenceis due to inhibition of angiogenesis (Guba et al., 2002). It has beenreported that the neurotrophic activities of immunophilin ligands areindependent of their immunosuppressive activity (Steiner et al., 1997)and that nerve growth stimulation is promoted by disruption of themature steroid receptor complex as outlined in the patent application WO01/03692. Side effects such as hyperlipidemia and thrombocytopenia aswell as potential teratogenic effects have been reported (Hentges etal., 2001; Kahan and Camardo, 2001).

The polyketide backbone of rapamycin is synthesised by head-to-tailcondensation of a total of seven propionate and seven acetate units to ashikimate derived cyclohexanecarboxylic acid starter unit by the verylarge, multifunctional proteins that comprise the Type I polyketidesynthase (rap PKS, Paiva et al., 1991). The L-lysine derived amino acid,pipecolic acid, is condensed via an amide linkage onto the last acetateof the polyketide backbone (Paiva et al., 1993) and is followed bylactonisation to form the macrocycle.

The nucleotide sequences for each of the three rapamycin PKS genes, theNRPS-encoding gene and the flanking late gene sequences and thecorresponding polypeptides, were identified by Aparicio et al., 1996,and Schwecke et al., 1995 and were deposited with the NCBI underaccession number X86780, and corrections to this sequence have recentlybeen published in WO 04/007709.

The first enzyme-free product of the rapamycin biosynthetic cluster hasbeen designated pre-rapamycin (WO 04/007709, Gregory et al., 2004).Production of the fully processed rapamycin requires additionalprocessing of the polyketide/NRPS core by the enzymes encoded by therapamycin late genes, RapJ, RapN, RapO, RapM, RapQ and RapI.

The pharmacologic actions of rapamycin characterised to date arebelieved to be mediated by the interaction with cytosolic receptorstermed FKBPs. The major intracellular rapamycin receptor in eukaryoticT-cells is FKBP12 (DiLella and Craig, 1991) and the resulting complexinteracts specifically with target proteins to inhibit the signaltransduction cascade of the cell.

The target of the rapamycin-FKBP12 complex has been identified in yeastas TOR (target of rapamycin) (Alarcon et al., 1999) and the mammalianprotein is known as FRAP (FKBP-rapamycin associated protein) or mTOR(mammalian target of rapamycin) (Brown et al., 1994).

A link between mTOR signalling and localized protein synthesis inneurons; its effect on the phosphorylation state of proteins involved intranslational control; the abundance of components of the translationmachinery at the transcriptional and translational levels; control ofamino acid permease activity and the coordination of the transcriptionof many enzymes involved in metabolic pathways have been described(Raught et al., 2001). Rapamycin sensitive signalling pathways alsoappear to play an important role in embryonic brain development,learning and memory formation (Tang et al., 2002). Research on TORproteins in yeast also revealed their roles in modulatingnutrient-sensitive signalling pathways (Hardwick et al., 1999).Similarly, mTOR has been identified as a direct target for the action ofprotein kinase B (akt) and of having a key role in insulin signalling(Shepherd et al., 1998; Navé et al., 1999). Mammalian TOR has also beenimplicated in the polarization of the actin cytoskeleton and theregulation of translational initiation (Alarcon et al., 1999).Phosphatidylinositol 3-kinases, such as mTOR, are functional in severalaspects of the pathogenesis of tumours such as cell-cycle progression,adhesion, cell survival and angiogenesis (Roymans and Slegers, 2001).

Pharmacokinetic studies of rapamycin and rapamycin analogues havedemonstrated the need for the development of novel rapamycin compoundsthat may be more stable in solution, more resistant to metabolic attackand/or have improved cell membrane permeability and decreased efflux andwhich therefore may exhibit improved oral bio-availability.

A range of synthesised rapamycin analogues using the chemicallyavailable sites of the molecule has been reported. The description ofthe following compounds was adapted to the numbering system of therapamycin molecule described in FIG. 1. Chemically available sites onthe molecule for derivatisation or replacement include C40 and C28hydroxyl groups (e.g. U.S. Pat. No. 5,665,772; U.S. Pat. No. 5,362,718),C39 and C16 methoxy groups (e.g. WO 96/41807; U.S. Pat. No. 5,728,710),C32, C26 and C9 keto groups (e.g. U.S. Pat. No. 5,378,836; U.S. Pat. No.5,138,051; U.S. Pat. No. 5,665,772). Hydrogenation at C17, C19 and/orC21, targeting the triene, resulted in retention of antifungal activitybut relative loss of immunosuppression (e.g. U.S. Pat. No. 5,391,730;U.S. Pat. No. 5,023,262). Significant improvements in the stability ofthe molecule (e.g. formation of oximes at C32, C40 and/or C28, U.S. Pat.No. 5,563,145, U.S. Pat. No. 5,446,048), resistance to metabolic attack(e.g. U.S. Pat. No. 5,912,253), bioavailability (e.g. U.S. Pat. No.5,221,670; U.S. Pat. No. 5,955,457; WO 98/04279) and the production ofprodrugs (e.g. U.S. Pat. No. 6,015,815; U.S. Pat. No. 5,432,183) havebeen achieved through derivatisation.

However, there remains a need for a greater range of rapamycinderivatives with improved metabolic stability, improved cell membranepermeability and/or a decreased rate of efflux. Such rapamycinderivatives would have great utility in the treatment of a wide range ofconditions. The present invention provides a range of39-desmethoxyrapamycin derivatives with improved metabolic stability,improved cell membrane permeability and/or a decreased rate of effluxand/or a different cell inhibitory profile to rapamycin. Such compoundsare useful in medicine, in particular for the treatment of cancer and/orB-cell malignancies, the induction or maintenance of immunosuppression,the treatment of transplantation rejection, graft vs. host disease,autoimmune disorders, diseases of inflammation, vascular disease andfibrotic diseases, the stimulation of neuronal regeneration or thetreatment of fungal infections.

SUMMARY OF THE INVENTION

The present invention provides 39-desmethoxy derivatives of rapamycin,methods for the preparation of these compounds, intermediates theretoand methods for the use of these compounds in medicine.

In its broadest aspect the present invention provides 39-desmethoxyderivatives of rapamycin characterised in that the 40-hydroxy positionis derivatised as a carboxylic acid ester, as an ether, as a phosphateester, as a phosphinate ester, as an acetal or as a glycosyl.

The metabolic stability, cell membrane permeability, efflux andbioavailability of the compounds of the invention may be tested as setout below.

When 39-desmethoxyrapamycin is derivatised as a carboxylic acid ester,as an ether or as an acetal the derivatising group preferably containsno more than 12 carbon atoms (especially 7 or fewer particularly 5 orfewer carbon atoms). Preferably it contains at least one functionalgroup (especially at least two functional groups) selected from—CF₂PO(OH)₂, —PO(OH)₂, —COOH, —OH and —NH₂ particularly selected from—COOH and —OH more particularly —OH.

When 39-desmethoxyrapamycin is derivatised as an acetal derived from aglycosyl group preferably each glycosyl is formed from a sugar or aglycoside which preferably contains no more than 12 carbon atoms(especially 7 or fewer, particularly 6 or fewer carbon atoms). Examplesinclude mono and disaccharides, particularly monosaccharides which form5 and 6 membered rings. Preferably it contains at least one functionalgroup (especially at least two function groups) selected from —COOH, —OHand —NH₂ particularly selected from —NH₂ and —OH more particularly —OH.

When 39-desmethoxyrapamycin is derivatised as a phosphate esterpreferably the alkyl groups contain no more than 4 carbon atoms.

When 39-desmethoxyrapamcyin is derivatised as a phosphinate esterpreferably the alkyl groups preferably contain no more than 4 carbonatoms, an example is the ester formed with phosphinic acid.

Specific examples of derivatising moieties are given below.

In a more specific aspect the present invention provides39-desmethoxyrapamycin derivatives according to formula (I) below, or apharmaceutically acceptable salt thereof:

wherein:

-   X represents bond or CH₂;-   R₁ represents a keto group or (H,H);-   R₂ represents OH or OMe;-   R₃ represents H, OH or OMe;-   R₄ and R₅ each independently represent H or OH;-   R₆ represents —R₇, —C(O)R₇, —(CH₂)₂—O—[CR₂₁R₂₂—O]_(a)—C(O)—R₂₃;    —CR₂₁R₂₂—O—C(O)—R₂₃; —POR₁₉R₂₀, —PO(OR₁₉)(OR₂₀) or Y—R₁₅;-   R₇ represents —(CR₈R₉)_(m)(CR₁₀R₁₁)_(p)CR₁₂R₁₃R₁₄;-   R₈ and R₉ each independently represent C1-C4 alkyl, C2-C4 alkenyl or    C2-C4 alkynyl, any of which groups may optionally be substituted    with —PO(OH)₂, —CF₂PO(OH)₂, —OH, —COOH or —NH₂; or R₈ and R₉ each    independently represent H, trifluoromethyl or F;-   R₁₀, R₁₁, R₁₂, R₁₃ and R₁₄ each independently represent C1-C4 alkyl,    C2-C4 alkenyl or C2-C4 alkynyl, any of which groups may optionally    be substituted with —PO(OH)₂, —CF₂PO(OH)₂, —OH, —COOH or —NH₂; or    R₁₀, R₁₁, R₁₂, R₁₃ and R₁₄ may be independently selected from H,    —(CR₈R₉)_(q)NH₂, —(CR₈R₉)_(q)OH, CF₃, F, COOH; or R₁₀ and R₁₁ or R₁₂    and R₁₃ or R₁₃ and R₁₄ may be taken together with the carbon to    which they are joined to form a C3-C6 cycloalkyl or a 3 to 6    membered heteroalkyl ring that contains one or more heteroatoms    selected from N, O and S and that is optionally, substituted with up    to 5 —(CR₈R₉)_(q)OH, —(CR₈R₉)_(q)NH₂ or COOH groups;-   Y=bond, —C(O)—O—; —(CH₂)₂—O—C(O)—O—;-   R₁₅ represents

-   R₁₆ are each independently H or OH;-   R₁₇ is independently selected from H, OH and NH₂;-   R₁₈ is independently selected from H, —CH₃, —CH₂OH and —COOH;-   provided however that no more than 2 groups selected from R₁₆, R₁₇    and R₁₈ represent H or CH₃;-   R₁₉ and R₂₀ each independently represent H or C1-C4 alkyl or R₁₉ and    R₂₀ together represent ═CH₂;-   R₂₁ is independently selected from H, CH₃;-   R₂₂ is independently selected from H, —CH₃, —CH═CH₂, —CH₂Cl, —CHCl₂,    —CCl₃, —CH(OH)Me, —CH₂OH, —CH₂CH₃, —CH(Cl)Me;-   R₂₃ is independently R₇, Y—R₁₅ or a 5 or 6 membered aryl or    heteroaryl ring optionally substituted with between one and three    groups selected from OH, F, Cl, Br, NO₂ and NH₂;-   a represents 0 or 1;-   m, p and q each independently represent an integer between 0-4;-   provided however that the R₇ moiety does not contain more than 12    carbon atoms and does contain at least one functional group selected    from —PO(OH)₂, —CF₂PO(OH)₂, —COOH, OH or NH₂; or a pharmaceutically    acceptable salt thereof.

The above structure shows a representative tautomer and the inventionembraces all tautomers of the compounds of formula (I) for example ketocompounds where enol compounds are illustrated and vice versa.

Unless particular stereoisomers are specifically indicated (e.g. by abolded or dashed bond at a relevant stereocentre in a structuralformula, by depiction of a double bond as having E or Z configuration ina structural formula, or by using stereochemistry-designatingnomenclature), all stereoisomers are included within the scope of theinvention as pure compounds as well as mixtures thereof. Unlessotherwise indicated, individual enantiomers, diastereomers, geometricalisomers, and combinations and mixtures thereof are all encompassed bythe present invention. Polymorphic crystalline forms and solvates andhydrates are also encompassed within the scope of this invention.

In a further aspect, the present invention provides39-desmethoxyrapamycin derivatives such as compounds of formula (I) or apharmaceutically acceptable salt thereof, for use as a pharmaceutical.

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example “an analogue” means one analogue or more than oneanalogue.

As used herein the term “analogue(s)” refers to chemical compounds thatare structurally similar to another but which differ slightly incomposition (as in the replacement of one atom by another or in thepresence or absence of a particular functional group).

In particular, the term “39-desmethoxyrapamycin analogue” refers to a39-desmethoxyrapamycin compound produced by the methods of WO2004/007709 and/or as shown by formula (II). These compounds are alsoreferred to as “parent compounds” and these terms are usedinterchangeably in the present application. In the present applicationthe term “39-desmethoxyrapamycin analogue” includes reference to39-desmethoxyrapamycin itself.

As used herein the term “derivative(s)” refers to chemical compoundsthat have been modified from their parent compound by semi-syntheticorganic chemistry.

In particular, the term “39-desmethoxyrapamycin derivative” refers to a39-desmethoxyrapamycin derivative according to formula (I) above, or apharmaceutically acceptable salt thereof, produced by semi-syntheticalteration of a 39-desmethoxyrapamycin analogue. These compounds arealso referred to as “compounds of the invention” or “39-desmethoxyderivatives of rapamycin” and these terms are used interchangeably inthe present application.

As used herein, the term “autoimmune disorder(s)” includes, withoutlimitation: systemic lupus erythrematosis (SLE), rheumatoid arthritis,myasthenia gravis and multiple sclerosis.

As used herein, the term “diseases of inflammation” includes, withoutlimitation: psoriasis, dermatitis, eczema, seborrhoea, inflammatorybowel disease (including but not limited to ulcerative colitis andCrohn's disease), pulmonary inflammation (including asthma, chronicobstructive pulmonary disease, emphysema, acute respiratory distresssyndrome and bronchitis), rheumatoid arthritis and eye uveitis.

As used herein, the term “cancer” refers to malignant growth of cells inskin or in body organs, for example but without limitation, breast,prostate, lung, kidney, pancreas, stomach or bowel. A cancer tends toinfiltrate into adjacent tissue and spread (metastasise) to distantorgans, for example to bone, liver, lung or the brain. As used hereinthe term cancer includes both metastatic tumour cell types, such as butnot limited to, melanoma, lymphoma, leukaemia, fibrosarcoma,rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such asbut not limited to, colorectal cancer, prostate cancer, small cell lungcancer and non-small cell lung cancer, breast cancer, pancreatic cancer,bladder cancer, renal cancer, gastric cancer, glioblastoma, primaryliver cancer and ovarian cancer.

As used herein the term “B-cell malignancies” includes a group ofdisorders that include chronic lymphocytic leukaemia (CLL), multiplemyeloma, and non-Hodgkin's lymphoma (NHL). They are neoplastic diseasesof the blood and blood forming organs. They cause bone marrow and immunesystem dysfunction, which renders the host highly susceptible toinfection and bleeding.

As used herein, the term “vascular disease” includes, withoutlimitation: hyperproliferative vascular disorders (e.g. restenosis andvascular occlusion), graft vascular atherosclerosis, cardiovasculardisease, cerebral vascular disease and peripheral vascular disease (e.g.coronary artery disease, arteriosclerosis, atherosclerosis,nonatheromatous arteriosclerosis or vascular wall damage).

As used herein the terms “neuronal regeneration” refers to thestimulation of neuronal cell growth and includes neurite outgrowth andfunctional recovery of neuronal cells. Diseases and disorders whereneuronal regeneration may be of significant therapeutic benefit include,but are not limited to, Alzheimer's disease, Parkinson's disease,Huntington's chorea, amyotrophic lateral sclerosis, trigeminalneuralgia, glossopharyngeal neuralgia, Bell's palsy, muscular dystrophy,stroke, progressive muscular atrophy, progressive bulbar inheritedmuscular atrophy, cervical spondylosis, Gullain-Barre syndrome,dementia, peripheral neuropathies and peripheral nerve damage, whethercaused by physical injury (e.g. spinal cord injury or trauma, sciatic orfacial nerve lesion or injury) or a disease state (e.g. diabetes).

As used herein the term “fibrotic diseases” refers to diseasesassociated with the excess production of the extracellular matrix andincludes (without limitation) sarcoidosis, keloids, glomerulonephritis,end stage renal disease, liver fibrosis (including but not limited tocirrhosis, alcohol liver disease and steato-heptatitis), chronic graftnephropathy, surgical adhesions, vasculopathy, cardiac fibrosis,pulmonary fibrosis (including but not limited to idiopathic pulmonaryfibrosis and cryptogenic fibrosing alveolitis), macular degeneration,retinal and vitreal retinopathy and chemotherapy or radiation-inducedfibrosis.

As used herein, the term “graft vs. host disease” refers to acomplication that is observed after allogeneic stem cell/bone marrowtransplant. It occurs when infection-fighting cells from the donorrecognize the patient's body as being different or foreign. Theseinfection-fighting cells then attack tissues in the patient's body justas if they were attacking an infection. Graft vs. host disease iscategorized as acute when it occurs within the first 100 days aftertransplantation and chronic if it occurs more than 100 days aftertransplantation. Tissues typically involved include the liver,gastrointestinal tract and skin. Chronic graft vs. host disease occursapproximately in 10-40 percent of patients after stem cell/bone marrowtransplant.

As used herein, the term “bioavailability” refers to the degree to whichor rate at which a drug or other substance is absorbed or becomesavailable at the site of biological activity after administration. Thisproperty is dependent upon a number of factors including the solubilityof the compound, rate of absorption in the gut, the extent of proteinbinding and metabolism etc. Various tests for bioavailability that wouldbe familiar to a person of skill in the art are described herein (seealso Trepanier et al, 1998, Gallant-Haidner et al, 2000).

The term “water solubility” as used in this application refers tosolubility in aqueous media, e.g. phosphate buffered saline (PBS) at pH7.4.

The pharmaceutically acceptable salts of compounds of the invention suchas the compounds of formula (I) include conventional salts formed frompharmaceutically acceptable inorganic or organic acids or bases as wellas quaternary ammonium acid addition salts. More specific examples ofsuitable acid salts include hydrochloric, hydrobromic, sulfuric,phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic,glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric,toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic,benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic andthe like. Other acids such as oxalic, while not in themselvespharmaceutically acceptable, may be useful in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable salts. More specific examples ofsuitable basic salts include sodium, lithium, potassium, magnesium,aluminium, calcium, zinc, N,N′-dibenzylethylenediamine, chloroprocaine,choline, diethanolamine, ethylenediamine, N-methylglucamine and procainesalts. References hereinafter to a compound according to the inventioninclude both compounds of formula (I) and their pharmaceuticallyacceptable salts.

Alkyl, alkenyl and alkynyl groups may be straight chain or branched.

Examples of C1-C4 alkyl groups include methyl, ethyl, n-propyl, i-propyland n-butyl.

Examples of C2-C4 alkenyl groups include ethenyl and 2-propenyl.

Examples of C2-4 alkynyl groups include ethynyl.

C3-C6 cycloalkyl groups refers to a cycloalkyl ring including 3-6 carbonatoms that may optionally be branched. Examples include cyclopropyl,cyclobutyl, methyl-cyclobutyl, cyclopentyl and cyclohexyl,

3 to 6 membered heteroalkyl rings containing one or more heteroatomsselected from N, O and S include rings containing one or twoheteroatoms, especially one heteroatom. Examples include furan, pyran,oxetane, oxirane, piperidine, pyrrolidine, azetidine, aziridine,thiirane, thiethane, thiophene, thiopyran and morpholine.

Example optional substituents for the 3 to 6 membered heteroalkyl ringsinclude —OH, —CH₂OH, NH₂, CH₂NH₂ and COOH. Typically the 3 to 6 memberedheteroalkyl rings may be unsubstituted or substituted by 1 or 2, e.g. 1substituent.

DESCRIPTION OF THE INVENTION

The present invention provides 39-desmethoxyrapamycin derivatives, asset out above, methods for the preparation of these compounds,intermediates thereto and methods for the use of these compounds inmedicine.

Preferably R₇ contains 7 or fewer especially 5 or fewer carbon atoms.

R₇ preferably contains at least one functional group selected from—PO(OH)₂, —OH, —COOH and —NH₂, more preferably —OH, —COOH or —NH₂,especially —COOH and OH, most especially OH. Preferably R₇ contains 2 ormore substituents, e.g. 2 —OH groups.

Suitably X represents CH₂;

Suitably a represents 0.

Suitably p represents 0 or 1.

Suitably m represents 0 or 1.

Suitably q represents 0, 1 or 2.

Suitably R₁₁ represents H. Suitably R₁₂ represents H.

Suitably R₁₃ represents H or OH.

When p represents 1, suitably R₁₀ represents Me, OH or CH₂OH.

When p represents 1, suitably R₁₁ represents Me, H or CH₂OH.

When m and p both represent 0, suitably R₁₂ and R₁₃ both represent H,R₁₄ represents —(CR₈R₉)_(q)—OH where q=0 or 1 and R₈ and R₉ bothrepresent H.

When p represents 1 and m represents 0, suitably R₁₀ and R₁₁ bothrepresent H, R₁₂ represents H, R₁₃ represents H, OH or NH₂, R₁₄represents —(CR₈R₉)_(q)—OH where q=0 or 1 and R₈ and R₉ both representH.

When R₆ represents —POR₁₅R₁₆ suitably R₁₅ and R₁₆ both represent CH₃ orboth represent CH₂CH₃.

Suitably R₆ represents the residue derived from forming an ester withhydroxylacetic acid, 3-hydroxy-2,2-dimethylpropionic acid,2,3-dihydroxypropionic acid, 3-hydroxy-2-hydroxymethylpropionic acid or2,2-bis(hydroxymethyl)propionic acid.

In one example set of compounds, R₆ represents: C(O)R₇

Preferably R₇ is the moiety formed by condensation of the macrocyclicalcohol with an acid selected from the list consisting of hydroxyaceticacid, 3-hydroxy-2,2,dimethylpropionic acid, 2,3-dihydroxypropionic acid,3-hydroxy-2-hydroxymethylpropionic acid and2,2-bis(hydroxymethyl)propionic acid, especially2,2-bis(hydroxymethyl)propionic acid.

When R₁₅ represents:

examples of this moiety include the moiety formed by forming an acetalwith (i) glucose (i.e. R₁₈ represents CH₂OH and each R₁₆ and R₁₇represents OH), e.g. D-glucose (ii) glucosamine (i.e. R₁₈ representsCH₂OH, each R₁₆ represents OH and R₁₇ represents NH₂) e.g.D-glucosamine, (iii) glucuronic acid (i.e. R₁₈ represents COOH and eachR₁₆ and R₁₇ represents OH) e.g. D-glucuronic acid and (iv) arabinose(i.e. R₁₈ represents H and each R₁₆ and R₁₇ represents OH) e.g.D-arabinose.

When R₁₅ represents:

examples of this moiety include the moiety formed by forming an acetalwith fructose (i.e. R₁₆ each represents OH), e.g. the residue ofD-fructose.

When R₁₅ represents:

examples of this moiety include the moiety formed by forming an esterwith glucuronic acid (i.e. each R₁₆ represents OH), e.g. the residue ofD-glucuronic acid.

In general, the compounds of the invention are prepared bysemi-synthetic derivatisation of a 39-desmethoxyrapamycin analogue offormula (II).

Thus a process for preparing a compound of formula (I) or apharmaceutically acceptable salt thereof comprises:

(a) reacting a 39-desmethoxyrapamycin analogue of formula (II):

where R_(A) represents H or (CH₂)₂—OH

or a protected derivative thereof, with a compound of formula (III):HO—R₆  (III)

or an activated derivative of R₆;

(b) converting a compound of formula (I) or a salt thereof to anothercompound of formula (I) or another pharmaceutically acceptable saltthereof; or

(c) deprotecting a protected compound of formula (I).

The term “activated derivative” as used above refers to (for example butwithout limitation): in the case of esters—carboxylic acids, acylhalides, mixed anhydrides, symmetrical anhydrides or carboxylic esters;in the case of ethers—alkyl halides, alkyl mesylates, alkyl triflates,alkyl tosylates or other suitably activated alkyl derivatives; in thecase of phosphates and phosphonates—chlorophosphates, dialkylcyanophosphates, dialkyl dialkylphosphoramidates or chlorophosphites; orin the case of acetals derived from glycosyl groups—using a glycosyldonor e.g. glycosyl halides, thioglycosides, 1-O-acyl glycosides, orthoesters, 1-O or 1-S carbonates, trichloroimidates, 4-pentenyl glycosides,glycosyl phosphate esters, 1-O-sulfonyls or 1-O-silylated glycosides.

In process (a), 39-desmethoxyrapamycin analogues of formula (II) may beprepared as described in WO 2004/007709 and as further set out in theexamples herein.

In addition to the specific methods and references provided herein aperson of skill in the art may also consult standard textbook referencesfor synthetic methods, including, but not limited to Vogel's textbook ofpractical organic chemistry (Furniss et al., 1989) and March's advancedorganic chemistry (Smith and March, 2001).

Additionally present hydroxyl groups can be protected by one of manystandard hydroxy protection strategies available to one skilled in theart. Hydroxyl groups may be protected by forming ethers, including, butnot limited to, substituted alkyl ethers, substituted benzyl ethers andsilyl ethers. Preferably a silyl ether, including, but not limited to,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl andt-butyldiphenylsilyl, ether is formed by reacting an activated form ofthe silane (including, but not limited to, silyl chloride or silyltriflate) with 39-desmethoxyrapamycin in the presence of a suitablebase. The protecting group could then be removed by either acidhydrolysis or fluoride assisted cleavage. 1,2-Diols may be protected asacetonides, based on the condensation of an acetone derivative. This maybe removed by acid catalysis.

The 39-desmethoxyrapamycin analogues of formula (II) may be used astemplates for further semi-synthesis (i.e. process (a)). The pendanthydroxyl group at C-40 can be functionalised by e.g. acylation,alkylation, glycosylation or phosphorylation via a number of synthetictransformations known to a person skilled in the art.

In process (a), when R₆ represents a moiety of formula —C(O)R₇ or Y—R₁₅where R₁₅ represents

and Y=bond, the formation of a hydroxy ester, or O-acylation, can bemediated by reaction of the hydroxyl group of the compounds of formula(II) with a corresponding carboxylic acid preferably in activated form,for example a compound of formula (IIIAi) or (IIIAii):

or with a compound of formula (IIIB):

where W is a group which activates a carboxylic acid to nucleophilicattack. Carboxylic acids can be activated by the formation of forexample but without limitation, acyl halides (e.g. W=Cl), mixedanhydrides (i.e. W=OC(O)R′), symmetrical anhydrides (W=OC(O)R₇) orcarboxylic esters (i.e. W=OR′).

Compounds of formula (IIIAi), (IIIAii) or (IIIB) can be prepared fromtheir commercially available carboxylic acids using standard methodsknown to a person of skill in the art, and in a specific aspectcompounds according to formula (IIIAi) wherein R₇ is—(CR₈R₉)_(m)(CR₁₀R₁₁)_(p)CR₁₂R₁₃R₁₄ may be prepared using methods asdescribed in U.S. Pat. No. 5,362,718, U.S. Pat. No. 5,665,772 or EP 0663 916.

Preferably a 39-desmethoxyrapamycin analogue is reacted in organic mediawith either an acid chloride or mixed anhydride in the presence of abase. Bases which may be used include, but are not limited to, pyridine,4,4-dimethylaminopyridine (DMAP), 2,6-lutidene,2,6-di-tert-butylpyridine, triethylamine, diisopropylethylamine, othertrialkylamines, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or1,5-diazabicyclo[4.3.0]non-5-ene (DBN). In specific examples describedherein, 39-desmethoxyrapamycin is reacted with a mixed anhydride in thepresence of DMAP.

In process (a), when R₆ represents a moiety of formula —C(O)R₇ or Y—R₁₅where R₁₅ represents

and Y=—C(O)O— or —(CH₂)₂—OC(O)O— the formation of these hydroxy esters,requires the reaction of the hydroxyl group of the compounds of formula(II) or a compound that is 40-O-(hydroxyethyl)-formula II with a reagentthat will form an activated carbonate such as a compound of formula IV

(IV), where T=bond or —O(CH₂)₂— and R₂₄ is an alkyl or aryl group,preferably an aryl group, especially para-nitrophenyl group.

The compound of formula IV can then react with a compound of formulaIII, to generate compounds with R₆ attached to the 40-hydroxyl group, or40-O-(hydroxyethyl) group via a carbonate linker (WO 2004/101583).

Likewise a 39-desmethoxyrapamycin analogue may be derivatised withdifferent hydroxy ethers at C-40, by reacting the 39-desmethoxyrapamycinanalogue with a suitably activated alkyl derivative of choice, to form a40-O-alkyl-39-desmethoxyrapamycin derivative. Activated alkyl groupsrefers to an alkyl group that has been activated by one of many methods,including, but not limited to, formation of alkyl halides (RCl, RI,RBr), alkyl mesylates (ROS(O)₂CH₃), alkyl triflates (ROS(O)₂CF₃), alkyltosylates (ROS(O)₂PhMe). The activated alkyl group would then be reactedwith a 39-desmethoxyrapamycin analogue in organic media in the presenceof a suitable base. Standard methods to optimise the reaction conditionsmay be employed by a person of skill in the art to avoid alkylation atother reactive positions.

Likewise a 39-desmethoxyrapamycin analogue may be phosphorylated, andafter deprotection of the phosphate esters it can yield a40-O-phospho-39-desmethoxyrapamycin derivative or a40-O-dialkylphospho-39-desmethoxyrapamycin derivative, and salts ofthese derivatives made by methods known to one skilled in the art.Phosphate esters can be formed directly, or indirectly via anO-phosphite (i.e. (R′O)₂POR) in which the trivalent phosphite isoxidised (preferably by the action of a peracid, such as but not limitednot mCPBA) to the pentavalent phosphate. Direct phosphorylation methodsinclude, but are not limited to, reaction of a 39-demethoxyrapamycinanalogue with a protected chlorophosphate (e.g. (BnO)₂P(O)Cl,(AlkylO)₂P(O)Cl), preferably in the presence of DMAP in organic media,or reaction of a 39-desmethoxyrapamycin analogue with phosphorusoxychloride (POCl₃), in the presence of a base such as triethylamine,followed by acid hydrolysis of the resultant O-dichlorophosphate (i.e.ROP(O)Cl₂), or coupling to a dialkyl cyanophosphate (WO 01/81355).Dialkyl or diaryl chlorophosphate may be generated in situ by thereaction of a dialkyl or diaryl phosphite (i.e. (RO)₂P(O)H) with carbontetrachloride in the presence of base. Methods of forming theO-phosphite (for oxidation to the O-phosphate) include, but are notlimited to, coupling a 39-desmethoxyrapamcyin analogue with a dialkyldialkylphosphoramidate (preferably dialkyldiisopropylphosphorylamidate), in the presence of base (preferablytetrazole), or coupling using a chlorophosphite in the presence of base(Evans et al., 1992). The choice of protecting group is important, ethyland methyl esters of phosphates are not readily hydrolysable underacidic or basic conditions. Preferably the protecting groups include,but are not limited to, benzyl esters (cleaved via sodiumiodide/chlorotrimethylsilane promoted hydrolysis, (WO 01/81355)) or2-cyanoethyl esters (cleaved via mild base catalysed cleavage).Similarly 40-O-dialkylphosphono-39-desmethoxyrapamycin derivatives canbe generated by reacting a 39-desmethoxyrapamycin analogue with asuitable activated (as described above) dialkylphosphonate ordialkylphosphite.

In process (a), when R₁₅ represents a moiety of formula

the formation of a glycosidic linkage, or O-glycosylation, can bemediated by reaction of the hydroxyl group with a corresponding glycosyldonor, preferably in activated form, (see Toshima and Tatsuta (1993))for example a compound of formula (IIIC):

or a compound of formula (IIID):

Using a ‘glycosyl donor’, including, but not limited to, glycosylhalides (Z=F, Cl, Br), thioglycosides (Z=SMe, Set, SPh, SPy, SCN),1-O-acyl glycosides (Z=OC(C)R), ortho esters (Z=OC(Me)(R)(O—C2 offormula (IIIC/IIID)), 1-O or 1-S carbonates (Z=OC(S)SMe,Z=OC(O)imidazole, Z=OC(S)imidazole, Z=SC(S)OEt), trichloroimidates(Z=OC(═NH)CCl₃), 4-pentenyl glycosides (Z=OCH₂CH₂CH₂CH═CH₂), phosphateesters (e.g. Z=OP(O)(OPh)₂), 1-O-sulfonyls (Z=tosyl), or 1-O-silylatedglycosides (Z=OTMS or OTBS), the 39-desmethoxyrapamycin analogue may beglycosylated in organic media, preferentially in the presence of anactivator (such as a Lewis acid or heavy metal salt, see Toshima andTatsuta, 1993)). The specific glycosyl donor used and the reactionconditions will determine whether an alpha or beta glycoside is formed.As before for acylation, any hydroxyl groups present in the parentcompound may be protected or masked such that using one equivalent ofglycosyl donor will result in 40-O-acylation. The remaining hydroxyls onthe glycosyl donor should be protected, as e.g. O-acetates, O-benzoates,1,2-acetonides, so a further deprotection will be necessary. Furthermore2-deoxyglycosyl donors such as glycals may be used (a reductive step isalso required) to prepare 2′-deoxy-39-desmethoxyrapamycin glycosides and2,6-dideoxyglycosyl donors such as 2,6-anhydro-2-thiosugars may be usedto prepare 2′,6′-dideoxy-39-desmethoxyrapamycin glycosides.

In process (b), salt formation and exchange may be performed byconventional methods known to a person of skill in the art.Interconversions of compounds of formula (I) may be performed by knownprocesses for example hydroxy and keto groups may be interconverted byoxidation/reduction as described elsewhere herein. Compounds of formula(I) in which R₆ represents —PO(OH)₂ may be prepared by phosphorylating acorresponding compound of formula (I) in which R₆ represents OH.Suitable conditions are provided elsewhere herein.

In processes (a) and (c), examples of protecting groups and the meansfor their removal can be found in T W Greene “Protective Groups inOrganic Synthesis” (J Wiley and Sons, 1991). Suitable hydroxylprotecting groups include alkyl (e.g. methyl), acetal (e.g. acetonide)and acyl (e.g. acetyl or benzoyl) which may be removed by hydrolysis,and arylalkyl (e.g. benzyl) which may be removed by catalytichydrolysis, or silyl ether, which may be removed by acidic hydrolysis orfluoride ion assisted cleavage.

In addition to process (a), 39-desmethoxyrapamycin analogues of formula(I) where R₆ represents R₇ can be synthesised by Lipase catalysedtransesterification. For example, but without limitation, a39-desmethoxyrapamycin analogue of formula (II) can be reacted with avinyl ester of formula (V) in the presence of lipase PS-C “Amano” IIunder the reaction conditions described by Gu et al (2005) and asfurther set out in the examples herein. This methodology is not limitedto the use of vinyl esters and the transesterification may be catalysedby other lipases or esterases.

Other compounds of the invention may be prepared by methods known per seor by methods analogous to those described above.

The novel 39-desmethoxyrapamycin derivatives are useful directly, and astemplates for further semi-synthesis or bioconversion, to producecompounds useful as immunosuppressants, antifungal agents, anticanceragents, anti-inflammatory agents, neuroregenerative agents or agents forthe treatment of transplantation rejection, graft vs. host disease,autoimmune disorders, vascular disease and/or fibrotic diseases. Methodsfor the semisynthetic derivatisation of rapamycin and analogues thereofare well known in the art and include (but are not limited to) thosemodifications described in e.g. U.S. Pat. No. 5,665,772; U.S. Pat. No.5,362,718, WO 96/41807; U.S. Pat. No. 5,728,710, U.S. Pat. No.5,378,836; U.S. Pat. No. 5,138,051; U.S. Pat. No. 5,665,772, U.S. Pat.No. 5,391,730; U.S. Pat. No. 5,023,262, U.S. Pat. No. 5,563,145, U.S.Pat. No. 5,446,048, U.S. Pat. No. 5,912,253, U.S. Pat. No. 5,221,670;U.S. Pat. No. 5,955,457; WO 98/04279, U.S. Pat. No. 6,015,815 and U.S.Pat. No. 5,432,183.

The above structures of intermediates (e.g. compounds of formula (II)may be subject to tautomerisation and where a representative tautomer isillustrated it will be understood that all tautomers for example ketocompounds where enol compounds are illustrated and vice versa areintended to be referred to.

In a further aspect, the present invention provides the use of the39-desmethoxyrapamycin derivatives of the invention in medicine. In afurther aspect the present invention provides for the use of39-desmethoxyrapamycin derivatives of the invention in the preparationof a medicament for the induction or maintenance of immunosuppression,the stimulation of neuronal regeneration or the treatment of cancer,B-cell malignancies, fungal infections, transplantation rejection, graftvs. host disease, autoimmune disorders, diseases of inflammationvascular disease and fibrotic diseases or agents for use in theregulation of wound healing.

Multi-Drug Resistance (MDR) is a significant problem in the treatment ofcancer and B-cell malignancies. It is the principle reason behind thedevelopment of drug resistance in many cancers (Persidis A, 1999). MDRis associated with increased level of adenosine triphosphate bindingcassette transporters (ABC transporters), in particular an increase inthe expression of the MDR1 gene which encodes for P-glycoprotein (P-gp)or the MRP1 gene which encodes MRP1. The level of MDR1 gene expressionvaries widely across different cancer-derived cell lines, in some celllines it is undetectable, whereas in others may show up to a 10 or100-fold increased expression relative to standard controls.

Therefore, a further aspect of the invention provides for the use of a39-desmethoxyrapamycin derivative of the invention in the treatment ofMDR cancers or B-cell malignancies. In a specific aspect the presentinvention provides for the use of 39-desmethoxyrapamycin derivatives inthe treatment of P-gp-expressing cancers or B-cell malignancies. In ayet more preferred embodiment the present invention provides for the useof a 39-desmethoxyrapamycin derivative of the invention in the treatmentof high P-gp expressing cancers or B-cell malignancies. Particularly,high P-gp expressing cancers or B-cell malignancies may have 2-fold,5-fold, 10-fold, 20-fold, 25-fold, 50-fold or 100-fold increasedexpression relative to control levels. Suitable controls are cells whichdo not express P-gp, which have a low expression level of P-gp or whichhave low MDR function, a person of skill in the art is aware of or canidentify such cell lines; by way of example (but without limitation)suitable cell lines include: MDA435/LCC6, SBC-3/CDDP, MCF7, NCI-H23,NCI-H522, A549/ATCC, EKVX, NCI-H226, NCI-H322M, NCI-H460, HOP-18,HOP-92, LXFL 529, DMS 114, DMS 273, HT29, HCC-2998, HCT-116, COLO 205,KM12, KM20L2, MDA-MB-231/ATCC, MDA-MB-435, MDA-N, BT-549, T-47D,OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, K-562, MOLT-4,HL-60(TB), RPMI-8226, SR, SN12C, RXF-631, 786-0, TK-10, LOX IMVI,MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, M14, UACC-62, UACC-257, PC-3,DU-145, SNB-19, SNB-75, SNB-78, U251, SF-268, SF-539, XF 498.

In an alternative aspect the present invention provides for the use of a39-desmethoxyrapamycin derivative of the invention in the preparation ofa medicament for use in the treatment of MDR cancers or B-cellmalignancies. In a specific aspect the present invention provides forthe use of a 39-desmethoxyrapamycin derivative of the invention in thepreparation of a medicament for use in the treatment of P-gp-expressingcancers or B-cell malignancies. In a yet more preferred embodiment thepresent invention provides for the use of a 39-desmethoxyrapamycinderivative in the preparation of a medicament for use in the treatmentof high P-gp expressing cancers or B-cell malignancies. Particularly,high P-gp expressing cancers or B-cell malignancies may have 2-fold,5-fold, 10-fold, 20-fold, 25-fold, 50-fold or 100-fold increasedexpression relative to control levels. Suitable controls are describedabove.

Methods for determining the expression level of P-gp in a sample arediscussed further herein.

Therefore, in a further aspect the present invention provides a methodfor the treatment of P-gp-expressing-cancers or B-cell malignanciescomprising administering a therapeutically effective amount of a39-desmethoxyrapamycin derivative of the invention. The expression levelof P-glycoprotein (P-gp) in a particular cancer type may be determinedby a person of skill in the art using techniques including but notlimited to real time RT-PCR (Szakács et al, 2004; Stein et al, 2002;Langmann et al; 2003, Alvarez et al, 1995, Boyd et al, 1995), byimmunohistochemistry (Stein et al, 2002) or using microarrays (Lee etal, 2003), these methods are provided as examples only, other suitablemethods will occur to a person of skill in the art.

One skilled in the art would be able by routine experimentation todetermine the ability of these compounds to inhibit fungal growth (e.g.Baker, H., et al., 1978; NCCLS Reference method for broth dilutionantifungal susceptibility testing for yeasts: Approved standard M27-A,17(9). 1997). Additionally, one skilled in the art would be able byroutine experimentation to determine the ability of these compounds toinhibit tumour cell growth, (see Dudkin, L., et al., 2001; Yu et al.2001). In a further aspect the compounds of this invention are usefulfor inducing immunosuppression, assays for determining a compound'sefficacy in these areas are well known to those of skill in the art, forexample but without limitation: Immunosuppressant activity—Warner, L.M., et al., 1992, Kahan et al. (1991) & Kahan & Camardo, 2001);Allografts—Fishbein, T. M., et al., 2002, Kirchner et al. 2000;Autoimmune/Inflammatory/Asthma—Carlson, R. P. et al., 1993, Powell, N.et al., 2001; Diabetes I—Rabinovitch, A. et al., 2002;Psoriasis—Reitamo, S. et al., 2001; Rheumatoid arthritis—Foey, A., etal., 2002; Fibrosis—Zhu, J. et al., 1999, Jain, S., et al., 2001,Gregory et al. 1993.

The ability of the 39-desmethoxyrapamycin derivatives of the inventionto induce immunosuppression may be demonstrated in standard tests usedfor this purpose. In a further aspect the 39-desmethoxyrapamycinderivatives of this invention are useful in relation to antifibrotic,neuroregenerative and anti-angiogenic mechanisms, one skilled in the artwould be able by routine experimentation to determine the ability ofthese compounds to prevent angiogenesis (e.g. Guba, M., et al., 2002).One of skill in the art would be able by routine experimentation todetermine the utility of these compounds to treat vascularhyperproliferative disease, for example in drug-eluting stents (e.g.Morice, M. C., et al., 2002). Additionally, one of skill in the artwould be able by routine experimentation to determine theneuroregenerative ability of these compounds (e.g. Myckatyn, T. M., etal., 2002, Steiner et al. 1997).

The present invention also provides a pharmaceutical compositioncomprising a 39-desmethoxyrapamycin derivative of the invention,together with a pharmaceutically acceptable carrier.

Rapamycin and related compounds that are or have been in clinicaltrials, such as CCI-779 and RAD001 have poor pharmacological profiles,poor water solubility and poor bioavailability. The present inventionprovides 39-desmethoxyrapamycin derivatives which have improvedproperties such as improved stability and/or increased cell membranepermeability. A person of skill in the art will be able to readilydetermine the solubility of a given compound of the invention usingstandard methods. A representative method is shown in the examplesherein.

Additionally, a person of skill in the art will be able to determine thepharmacokinetics and bioavailability of a compound of the inventionusing in vivo and in vitro methods known to a person of skill in theart, including but not limited to those described below and in theexamples, alternative assays are well known to a person of skill in theart including but not limited to those described below and inGallant-Haidner et al, 2000 and Trepanier et al, 1998 and referencestherein. The bioavailability of a compound is determined by a number offactors, (e.g. water solubility, rate of absorption in the gut, theextent of protein binding and metabolism) each of which may bedetermined by in vitro tests as described below, it will be appreciatedby a person of skill in the art that an improvement in one or more ofthese factors will lead to an improvement in the bioavailability of acompound. Alternatively, the bioavailability of a compound may bemeasured using in vivo methods as described in more detail below.

Caco-2 Permeation Assay

Confluent Caco-2 cells (Li, A. P., 1992; Grass, G. M., et al., 1992,Volpe, D. A., et al., 2001) in a 24 well Corning Costar Transwell formatmay be used, e.g. as provided by In Vitro Technologies Inc. (IVT Inc.,Baltimore, Md., USA). The apical chamber contains 0.15 mL Hank'sbalanced buffer solution (HBBS) pH 7.4, 1% DMSO, 0.1 mM Lucifer Yellow.The basal chamber contains 0.6 mL HBBS pH 7.4, 1% DMSO. Controls andtests are then incubated at 37° C. in a humidified incubator and shakenat 130 rpm for 1 h. Lucifer Yellow permeates via the paracellular(between the tight junctions) route only, a high Apparent Permeability(P_(app)) for Lucifer Yellow indicates cellular damage during assay andall such wells were rejected. Propranolol (good passive permeation withno known transporter effects) & acebutalol (poor passive permeationattenuated by active efflux by P-glycoprotein) are used as referencecompounds. Compounds may be tested in a uni- and bi-directional formatby applying compound to the apical or basal chamber (at 0.01 mM).Compounds in the apical or basal chambers are analysed by HPLC-MS.Results are expressed as Apparent Permeability, P_(app), (nm/s) and asthe Flux Ratio (A to B versus B to A).

${{Papp}\mspace{11mu}\left( {{nm}\text{/}s} \right)} = {\frac{{Volume}\mspace{14mu}{Acceptor}}{{Area} \times \lbrack{donor}\rbrack} \times \frac{\Delta\lbrack{acceptor}\rbrack}{\Delta\;{time}}}$

-   -   Volume Acceptor: 0.6 mL (A>B) and 0.15 mL (B>A)    -   Area of monolayer: 0.33 cm²    -   Δtime: 60 min

A positive value for the Flux Ratio indicates active efflux from theapical surface of the cells.

Human Liver Microsomal (HLM) Stability Assay

Liver homogenates provide a measure of a compounds inherentvulnerability to Phase I (oxidative) enzymes, including CYP450s (e.g.CYP2C8, CYP2D6, CYP1A, CYP3A4, CYP2E1), esterases, amidases and flavinmonooxygenases (FMOs).

The half life (T½) of test compounds can be determined, on exposure toHuman Liver Microsomes, by monitoring their disappearance over time byLC-MS. Compounds at 0.001 mM are incubated at for 40 min at 37° C., 0.1M Tris-HCl, pH 7.4 with human microsomal sub-cellular fraction of liverat 0.25 mg/mL protein and saturating levels of NADPH as co-factor. Attimed intervals, acetonitrile is added to test samples to precipitateprotein and stop metabolism. Samples are centrifuged and analysed forparent compound by HPLC-MS.

In Vivo Bioavailability Assays

In vivo assays may also be used to measure the bioavailability of acompound (see e.g. Crowe et al, 1999). Generally, a compound isadministered to a test animal (e.g. mouse or rat) both intraperitoneally(i.p.) or intravenously (i.v.) and orally (p.o.) and blood samples aretaken at regular intervals to examine how the plasma concentration ofthe drug varies over time. The time course of plasma concentration overtime can be used to calculate the absolute bioavailability of thecompound as a percentage using standard models. An example of a typicalprotocol is described below.

Mice are dosed with 3 mg/kg of the compound of the invention or theparent compound i.v. or 10 mg/kg of a compound of the invention of theparent compound p.o. Blood samples are taken at 5 minute, 15 minute, 1h, 4 h and 24 h intervals and the concentration of the compound of theinvention or parent compound in the sample is determined via HPLC. Thetime-course of plasma concentrations can then be used to derive keyparameters such as the area under the plasma concentration-time curve(AUC—which is directly proportional to the total amount of unchangeddrug that reaches the systemic circulation), the maximum (peak) plasmadrug concentration, the time at which maximum plasma drug concentrationoccurs (peak time), additional factors which are used in the accuratedetermination of bioavailability include: the compound's terminal halflife, total body clearance, steady-state volume of distribution and F %.These parameters are then analysed by non-compartmental or compartmentalmethods to give a calculated percentage bioavailability, for an exampleof this type of method see Gallant-Haidner et al, 2000 and Trepanier etal, 1998 and references therein, and references therein.

The aforementioned 39-desmethoxyrapamycin derivatives of the inventionor a formulation thereof may be administered by any conventional methodfor example but without limitation they may be administeredparenterally, orally, topically (including buccal, sublingual ortransdermal), via a medical device (e.g. a stent), by inhalation or viainjection (subcutaneous or intramuscular). The treatment may consist ofa single dose or a plurality of doses over a period of time.

Whilst it is possible for a compound of the invention to be administeredalone, it is preferable to present it as a pharmaceutical formulation,together with one or more acceptable carriers. The carrier(s) must be“acceptable” in the sense of being compatible with the compound of theinvention and not deleterious to the recipients thereof. Examples ofsuitable carriers are described in more detail below.

The 39-desmethoxyrapamycin derivatives of the invention may beadministered alone or in combination with other therapeutic agents,co-administration of two (or more) agents allows for significantly lowerdoses of each to be used, thereby reducing the side effects seen.

In one embodiment, a 39-desmethoxyrapamycin derivative isco-administered with another therapeutic agent for the induction ormaintenance of immunosuppression, for the treatment of transplantationrejection, graft vs. host disease, autoimmune disorders or diseases ofinflammation preferred agents include, but are not limited to,immunoregulatory agents e.g. azathioprine, corticosteroids,cyclophosphamide, cyclosporin A, FK506, Mycophenolate Mofetil, OKT-3 andATG.

In an alternative embodiment, a 39-desmethoxyrapamycin derivative isco-administered with another therapeutic agent for the treatment ofcancer or B-cell malignancies preferred agents include, but are notlimited to, methotrexate, leukovorin, adriamycin, prenisone, bleomycin,cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine,vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrolacetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g.Herceptin™), capecitabine, raloxifene hydrochloride, EGFR inhibitors(e.g. Iressa®, Tarceva™, Erbitux™), VEGF inhibitors (e.g. Avastin™),proteasome inhibitors (e.g. Velcade™), Glivec® or hsp90 inhibitors (e.g.17-AAG). Additionally, a 39-desmethoxyrapamycin derivative may beadministered in combination with other therapies including, but notlimited to, radiotherapy or surgery.

In one embodiment, a 39-desmethoxyrapamycin derivative isco-administered with another therapeutic agent for the treatment ofvascular disease, preferred agents include, but are not limited to, ACEinhibitors, angiotensin II receptor antagonists, fibric acidderivatives, HMG-CoA reductase inhibitors, beta adrenergic blockingagents, calcium channel blockers, antioxidants, anticoagulants andplatelet inhibitors (e.g. Plavix™).

In one embodiment, a 39-desmethoxyrapamycin derivative isco-administered with another therapeutic agent for the stimulation ofneuronal regeneration, preferred agents include, but are not limited to,neurotrophic factors e.g. nerve growth factor, glial derived growthfactor, brain derived growth factor, ciliary neurotrophic factor andneurotrophin-3.

In one embodiment, a 39-desmethoxyrapamycin derivative isco-administered with another therapeutic agent for the treatment offungal infections; preferred agents include, but are not limited to,amphotericin B, flucytosine, echinocandins (e.g. caspofungin,anidulafungin or micafungin), griseofulvin, an imidazole or a triazoleantifungal agent (e.g. clotrimazole, miconazole, ketoconazole,econazole, butoconazole, oxiconazole, terconazole, itraconazole,fluconazole or voriconazole).

By co-administration is included any means of delivering two or moretherapeutic agents to the patient as part of the same treatment regime,as will be apparent to the skilled person. Whilst the two or more agentsmay be administered simultaneously in a single formulation this is notessential. The agents may administered in different formulations and atdifferent times.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the activeingredient (compound of the invention) with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

The 39-desmethoxyrapamycin derivatives of the invention will normally beadministered orally or by any parenteral route, in the form of apharmaceutical formulation comprising the active ingredient, optionallyin the form of a non-toxic organic, or inorganic, acid, or base,addition salt, in a pharmaceutically acceptable dosage form. Dependingupon the disorder and patient to be treated, as well as the route ofadministration, the compositions may be administered at varying doses.

For example, the compounds of the invention can be administered orally,buccally or sublingually in the form of tablets, capsules, ovules,elixirs, solutions or suspensions, which may contain flavouring orcolouring agents, for immediate-, delayed- or controlled-releaseapplications.

Solutions or suspensions of 39-desmethoxyrapamycin derivatives suitablefor oral administration may also contain excipients e.g.N,N-dimethylacetamide, dispersants e.g. polysorbate 80, surfactants, andsolubilisers, e.g. polyethylene glycol, Phosal 50 PG (which consists ofphosphatidylcholine, soya-fatty acids, ethanol, mono/diglycerides,propylene glycol and ascorbyl palmitate),

Such tablets may contain excipients such as microcrystalline cellulose,lactose (e.g. lactose monohydrate or lactose anyhydrous), sodiumcitrate, calcium carbonate, dibasic calcium phosphate and glycine,disintegrants such as starch (preferably corn, potato or tapiocastarch), sodium starch glycollate, croscarmellose sodium and certaincomplex silicates, and granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),macrogol 8000, sucrose, gelatin and acacia. Additionally, lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate andtalc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surface-active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provideslow or controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providedesired release profile.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, impregnated dressings, sprays, aerosols oroils, transdermal devices, dusting powders, and the like. Thesecompositions may be prepared via conventional methods containing theactive agent. Thus, they may also comprise compatible conventionalcarriers and additives, such as preservatives, solvents to assist drugpenetration, emollient in creams or ointments and ethanol or oleylalcohol for lotions. Such carriers may be present as from about 1% up toabout 98% of the composition. More usually they will form up to about80% of the composition. As an illustration only, a cream or ointment isprepared by mixing sufficient quantities of hydrophilic material andwater, containing from about 5-10% by weight of the compound, insufficient quantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active agent may be delivered from the patch byiontophoresis.

For applications to external tissues, for example the mouth and skin,the compositions are preferably applied as a topical ointment or cream.When formulated in an ointment, the active agent may be employed witheither a paraffinic or a water-miscible ointment base.

Alternatively, the active agent may be formulated in a cream with anoil-in-water cream base or a water-in-oil base.

For parenteral administration, fluid unit dosage forms are preparedutilizing the active ingredient and a sterile vehicle, for example butwithout limitation water, alcohols, polyols, glycerine and vegetableoils, water being preferred. The active ingredient, depending on thevehicle and concentration used, can be either suspended or dissolved inthe vehicle. In preparing solutions the active ingredient can bedissolved in water for injection and filter sterilised before fillinginto a suitable vial or ampoule and sealing.

Advantageously, agents such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum. The dry lyophilized powder is thensealed in the vial and an accompanying vial of water for injection maybe supplied to reconstitute the liquid prior to use.

Parenteral suspensions are prepared in substantially the same manner assolutions, except that the active ingredient is suspended in the vehicleinstead of being dissolved and sterilization cannot be accomplished byfiltration. The active ingredient can be sterilised by exposure toethylene oxide before suspending in the sterile vehicle. Advantageously,a surfactant or wetting agent is included in the composition tofacilitate uniform distribution of the active ingredient.

The compounds of the invention may also be administered using medicaldevices known in the art. For example, in one embodiment, apharmaceutical composition of the invention can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. No. 5,399,163; U.S. Pat. No. 5,383,851; U.S. Pat. No.5,312,335; U.S. Pat. No. 5,064,413; U.S. Pat. No. 4,941,880; U.S. Pat.No. 4,790,824; or U.S. Pat. No. 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicamentsthrough the skin; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. In a specific embodiment the39-desmethoxyrapamycin derivative may be administered using adrug-eluting stent, for example corresponding to those described in WO01/87263 and related publications or those described by Perin (Perin, EC, 2005). Many other such implants, delivery systems, and modules areknown to those skilled in the art.

The dosage to be administered of a 39-desmethoxyrapamycin derivative ofthe invention will vary according to the particular compound, thedisease involved, the subject, and the nature and severity of thedisease and the physical condition of the subject, and the selectedroute of administration. The appropriate dosage can be readilydetermined by a person skilled in the art.

The compositions may contain from 0.1% by weight, preferably from 5-60%,more preferably from 10-30% by weight, of a compound of invention,depending on the method of administration.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of a compound of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and the ageand condition of the particular subject being treated, and that aphysician will ultimately determine appropriate dosages to be used. Thisdosage may be repeated as often as appropriate. If side effects developthe amount and/or frequency of the dosage can be altered or reduced, inaccordance with normal clinical practice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows the structure of rapamycin

FIG. 2: shows the fragmentation pathway for 39-desmethoxyrapamycin

FIG. 3: shows the fragmentation pathway for39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin.

FIG. 4: shows the fragmentation pathway for39-desmethoxy-40-O-[2-hydroxyethyl3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin

FIG. 5: shows the fragmentation pathway for27-O-desmethyl-39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin

FIG. 6: shows the mTOR inhibitory activity of39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin (A—filledtriangles) and 39-desmethoxy-40-O-(2-hydroxy)ethyl rapamycin (B—filledtriangles) compared to rapamycin (filled squares).

EXAMPLES

General Methods and Materials

Materials

All reagents were obtained from commercial sources, and used withoutfurther purification unless stated otherwise.

Culture

S. hygroscopicus MG2-10 [IJMNOQLhis] (WO 04/007709; Gregory et al.,2004) was maintained on medium 1 agar plates (see below) at 28° C. Sporestocks were prepared after growth on medium 1, preserved in 20% w/vglycerol: 10% w/v lactose in distilled water and stored at −80° C.Vegetative cultures were prepared by inoculating 0.1 mL of frozen stockinto 50 mL medium 2 (see below) in 250 mL flask. The culture wasincubated for 36 to 48 hours at 28° C., 300 rpm.

Production Method:

Vegetative cultures were inoculated at 2.5-5% v/v into medium 3.Cultivation was carried out for 6-7 days, 26° C., 300 rpm.

Feeding Procedure:

The feeding/addition of the selected carboxylic acid was carried out24-48 hours after inoculation and was fed at 1-2 mM unless statedotherwise.

Medium 1:

component Source Catalogue # Per L Corn steep powder Sigma C-8160 2.5 gYeast extract Difco 0127-17 3 g Calcium carbonate Sigma C5929 3 g Ironsulphate Sigma F8633 0.3 g BACTO agar Difco 2140-10 20 g Wheat starchSigma S2760 10 g Water to 1 L

The media was then sterilised by autoclaving 121° C., 20 min.

Medium 2: RapV7 Seed Medium

Component Per L Toasted Nutrisoy (ADM Ingredients Ltd) 5 g Avedex W80dextrin (Deymer Ingredients Ltd) 35 g Corn Steep Solids (Sigma) 4 gGlucose 10 g (NH₄)₂SO₄ 2 g Lactic acid (80%) 1.6 mL CaCO₃(Caltec) 7 gAdjust pH to 7.5 with 1 M NaOH.

The media was then sterilised by autoclaving 121° C., 20 min.

After sterilisation 0.16 mL of 40% glucose is added to each 7 mL ofmedia.

Medium 3: MD6 Medium (Fermentation Medium)

Component Per L Toasted Nutrisoy (ADM Ingredients Ltd) 30 g Corn starch(Sigma) 30 g Avedex W80 dextrin (Deymer Ingredients Ltd) 19 g Yeast(Allinson) 3 g Corn Steep Solids (Sigma) 1 g KH₂PO₄ 2.5 g K₂HPO₄ 2.5 g(NH₄)₂SO₄ 10 g NaCl 5 g CaCO₃(Caltec) 10 g MnCl₂•4H₂O 10 mg MgSO₄•7H₂O2.5 mg FeSO₄•7H₂O 120 mg ZnSO₄•7H₂O 50 mg MES (2-morpholinoethanesulphuric acid monohydrate) 21.2 g pH is corrected to 6.0 with 1 M NaOH

Before sterilization 0.4 mL of Sigma α-amylase (BAN 250) was added to 1L of medium.

Medium was sterilised for 20 min at 121° C.

After sterilisation 0.35 mL of sterile 40% fructose and 0.10 mL ofL-lysine (140 mg/mL in water, filter-sterilised) was added to each 7 mL.

Medium 4: Rap V7a Seed Medium

Component Per L Toasted Nutrisoy (ADM Ingredients Ltd) 5 g Avedex W80dextrin (Deymer Ingredients Ltd) 35 g Corn Steep Solids (Sigma) 4 g(NH₄)₂SO₄ 2 g Lactic acid (80%) 1.6 mL CaCO₃(Caltec) 7 g Adjust pH to7.5 with 1 M NaOH.

The media was then sterilised by autoclaving 121° C., 20 min.

Medium 5: MD6/5-1 Medium (Fermentation Medium)

Component Per L Toasted Nutrisoy (ADM Ingredients Ltd) 15 g Avedex W80dextrin (Deymer Ingredients Ltd) 50 g Yeast (Allinson) 3 g Corn SteepSolids (Sigma) 1 g KH₂PO₄ 2.5 g K₂HPO₄ 2.5 g (NH₄)₂SO₄ 10 g NaCl 13 gCaCO₃(Caltec) 10 g MnCl₂ 4H₂O 3.5 mg MgSO₄ 7H₂O 15 mg FeSO₄ 7H₂O 150 mgZnSO₄ 7H₂O 60 mg SAG 471 0.1 ml

Medium was sterilised for 30 min at 121° C.

After sterilisation 15 g of Fructose per L was added.

After 48 h 0.5 g/L of L-lysine was added.

Analytical Methods

Method A

Injection volume: 0.005-0.1 mL (as required depending on sensitivity).HPLC was performed on Agilent “Spherisorb” “Rapid Resolution” cartridgesSB C8, 3 micron, 30 mm×2.1 mm, running a mobile phase of:

Mobile phase A: 0.01% Formic acid in pure water Mobile phase B: 0.01%Formic acid in Acetonitrile Flow rate: 1 mL/minute.Linear gradient was used, from 5% B at 0 min to 95% B at 2.5 min holdingat 95% B until 4 min returning to 5% B until next cycle. Detection wasby UV absorbance at 254 nm and/or by mass spectrometry electrosprayionisation (positive or negative) using a Micromass Quattro-Microinstrument.Method B

Injection volume: 0.02 mL. HPLC was performed on 3 micron BDS C18Hypersil (ThermoHypersil-Keystone Ltd) column, 150×4.6 mm, maintained at50° C., running a mobile phase of:

Mobile phase A: Acetonitrile (100 mL), trifluoracetic acid (1 mL), 1 Mammonium acetate (10 mL) made up to 1 L with deionised water. Mobilephase B: Deionised water (100 mL), trifluoracetic acid (1 mL), 1Mammonium acetate (10 mL) made up to 1 L with acetonitrile. Flow rate 1mL/minute.A linear gradient from 55% B-95% B was used over 10 minutes, followed by2 minutes at 95% B, 0.5 minutes to 55% B and a further 2.5 minutes at55% B. Compound detection was by UV absorbance at 280 nm.Method C

The HPLC system comprised an Agilent HP 1100 and was performed on 3micron BDS C18 Hypersil (ThermoHypersil-Keystone Ltd) column, 150×4.6mm, maintained at 40° C., running a mobile phase of:

Mobile phase A: deionised water. Mobile phase B: acetonitrile. Flow rate1 mL/minute.The system was coupled to a Bruker Daltonics Esquire3000 electrospraymass spectrometer. Positive negative switching was used over a scanrange of 500 to 1000 Dalton.A linear gradient from 55% B-95% B was used over 10 minutes, followed by2 minutes at 95% B, 0.5 minutes to 55% B and a further 2.5 minutes at55% B.Synthetic Methods

All reactions were carried out under anhydrous conditions unless statedotherwise using commercially available dried solvents. Reactions weremonitored by LC-UV-MS, on an Agilent 1100 HPLC coupled to a BrukerDaltonics Esquire3000+ mass spectrometer equipped with an electrospraysource. Separation was achieved over a Phenomenex Hyperclone column, BDSC₁₈ 3u (150×4.6 mm) at 1 mL/min, with a linear gradient ofwater:acetonitrile v:v 30:70 to 100% acetonitrile over 10 min followedby an isocratic period of 5 min at 100% acetonitrile.

NMR spectra were recorded in CDCl₃ and δ_(H) and δ_(C) chemical shiftsare referenced to the solvent (7.26 ppm and 77.0 ppm respectively).Since 39-desmethoxyrapamycin and its derivatives exist as a mixture ofconformers all assignments correspond to the major conformer only.

In Vitro Bioassay for Anticancer Activity

In vitro evaluation of compounds for anticancer activity in a panel of12 human tumour cell lines in a monolayer proliferation assay wascarried out at the Oncotest Testing Facility, Institute for ExperimentalOncology, Oncotest GmbH, Freiburg. The characteristics of the 12selected cell lines is summarised in Table 1.

TABLE 1 Test cell lines # Cell line Characteristics 1 MCF-7 Breast, NCIstandard 2 MDA-MB-231 Breast - PTEN positive, resistant to 17-AAG 3MDA-MB-468 Breast - PTEN negative, resistant to 17-AAG 4 NCI-H460 Lung,NCI standard 5 SF-268 CNS, NCI standard 6 OVCAR-3 Ovarian - p85 mutated.AKT amplified. 7 A498 Renal, high MDR expression, 8 GXF 251L Gastric 9MEXF 394NL Melanoma 10 UXF 1138L Uterus 11 LNCAP Prostate - PTENnegative 12 DU145 Prostate - PTEN positive

The Oncotest cell lines were established from human tumor xenografts asdescribed by Roth et al. 1999. The origin of the donor xenografts wasdescribed by Fiebig et al. 1999. Other cell lines were either obtainedfrom the NCI (H460, SF-268, OVCAR-3, DU145, MDA-MB-231, MDA-MB-468) orpurchased from DSMZ, Braunschweig, Germany (LNCAP).

All cell lines, unless otherwise specified, are grown at 37° C. in ahumidified atmosphere (95% air, 5% CO₂) in a ‘ready-mix’ mediumcontaining RPMI 1640 medium, 10% fetal calf serum, and 0.1 mg/mLgentamicin (PAA, Cölbe, Germany).

Monolayer Assay—Brief Description of Protocol 1:

A modified propidium iodide assay was used to assess the effects of thetest compound(s) on the growth of twelve human tumor cell lines (Dengleret al., (1995)).

Briefly, cells were harvested from exponential phase cultures bytrypsinization, counted and plated in 96 well flat-bottomed microtitreplates at a cell density dependent on the cell line (5-10,000 viablecells/well). After 24 h recovery to allow the cells to resumeexponential growth, 0.01 mL of culture medium (6 control wells perplate) or culture medium containing macbecin are added to the wells.Each concentration is plated in triplicate. Compounds are applied in twoconcentrations (0.001 μM and 0.01 μM). Following 4 days of continuousexposure, cell culture medium with or without test compound is replacedby 0.2 mL of an aqueous propidium iodide (PI) solution (7 mg/L). Tomeasure the proportion of living cells, cells are permeabilized byfreezing the plates. After thawing the plates, fluorescence is measuredusing the Cytofluor 4000 microplate reader (excitation 530 nm, emission620 nm), giving a direct relationship to the total number of viablecells.

Growth inhibition is expressed as treated/control×100 (% T/C). Foractive compounds, IC₅₀ & IC₇₀ values were estimated by plotting compoundconcentration versus cell viability.

Example 1 Fermentation and Isolation of 39-desmethoxyrapamycin

39-desmethoxyrapamycin was produced by growing cultures of S.hygroscopicus MG2-[IJMNOQLhis] and feeding with cyclohexanecarboxylicacid (CHCA) as described below.

Liquid Culture

A vegetative culture of S. hygroscopicus MG2-10 [IJMNOQLhis] wascultivated as described in Materials & Methods. Production cultures wereinoculated with vegetative culture at 0.5 mL into 7 mL medium 3 in 50 mLtubes. Cultivation was carried out for 7 days, 26° C., 300 rpm. Onemilliliter samples were extracted 1:1 acetonitrile with shaking for 30min, centrifuged 10 min, 13,000 rpm and analysed and quantifiedaccording to analysis Method B (see Materials & Methods). Confirmationof product was determined by mass spectrometry using analysis Method C(see Materials & Methods).

The observed rapamycin analogue was proposed to be the desired39-desmethoxyrapamycin on the basis of the analytical data discussedunder characterisation below.

Fermentation

A primary vegetative culture in Medium 4 of S. hygroscopicus MG2-10[IJMNOQLhis] was cultivated essentially as described in Materials &Methods. A secondary vegetative culture in Medium 4 was inoculated at10% v/v, 28° C., 250 rpm, for 24 h. Vegetative cultures were inoculatedat 5% v/v into medium 5 (see Materials & Methods) in a 20 L fermenter.Cultivation was carried out for 6 days at 26° C., 0.5 vvm.≧30% dissolvedoxygen was maintained by altering the impeller tip speed, minimum tipspeed of 1.18 ms⁻¹ maximum tip speed of 2.75 ms⁻¹. The feeding ofcyclohexanecarboxylic acid was carried out at 24 and 48 hours afterinoculation to give a final concentration of 2 mM.

Extraction and Purification

The fermentation broth (30 L) was stirred with an equal volume ofmethanol for 2 hours and then centrifuged to pellet the cells (10 min,3500 rpm). The supernatant was stirred with Diaion® HP20 resin (43 g/L)for 1 hour and then filtered. The resin was washed batchwise withacetone to strip off the rapamycin analogue and the solvent was removedin vacuo. The aqueous concentrate was then diluted to 2 L with water andextracted with ethyl acetate (3×2 L). The solvent was removed in vacuoto give a brown oil (20.5 g).

The extract was dissolved in acetone, dried onto silica, applied to asilica column (6×6.5 cm diameter) and eluted with a stepwise gradient ofacetone/hexane (20%-40%). The rapamycin analogue-containing fractionswere pooled and the solvent removed in vacuo. The residue (2.6 g) wasfurther chromatographed (in three batches) over Sephadex LH20, elutingwith 10:10:1 chloroform/heptane/ethanol. The semipurified rapamycinanalogue (1.7 g) was purified by reverse phase (C18) preparative HPLCusing a Gilson HPLC, eluting a Phenomenex 21.2×250 mm Luna 5 μm C18 BDScolumn with 21 mL/min of 65% acetonitrile/water. The most pure fractions(identified by analytical HPLC, Method B) were combined and the solventremoved in vacuo to give 39-desmethoxyrapamycin (563 mg).

Characterisation

The ¹H NMR spectrum of 39-desmethoxyrapamycin was equivalent to that ofa standard (P. Lowden, Ph.D. Dissertation, University of Cambridge,1997). ¹³C-NMR (125 MHz), δ_(C) (ppm): 215.75, 208.27, 169.19, 166.71,140.13, 135.94, 133.61, 130.10, 129.62, 126.80, 126.33, 98.42, 84.77,84.37, 75.85, 70.91, 67.10, 59.44, 55.82, 51.21, 46.50, 44.17, 41.39,40.70, 40.16, 38.74, 38.37, 35.44, 35.26, 35.08, 33.78, 33.64, 33.04,32.37, 31.22, 30.41, 27.24, 27.02, 25.27, 21.48, 20.58, 16.24, 15.95,15.78, 13.74, 13.00, 10.12.

LCMS and LCMS^(n) analysis of culture extracts showed that the m/z ratiofor the novel rapamycin analogue is 30 atomic mass units lower than thatfor rapamycin, consistent with the absence of a methoxy group. Ionsobserved: [M−H]⁻ 882.3, [M+NH₄]+901.4, [M+Na]⁺ 906.2, [M+K]⁺ 922.2.Fragmentation of the sodium adduct gave the predicted ions for39-desmethoxyrapamycin following a previously identified fragmentationpathway (FIG. 2) (J. A. Reather, Ph.D. Dissertation, University ofCambridge, 2000). This mass spectrometry fragmentation data narrows theregion of the novel rapamycin analogue where the loss of a methoxy hasoccurred to the fragment C28-C42 that contains the cyclohexyl moiety.This mass spectrometry fragmentation data is entirely consistent with39-desmethoxyrapamycin.

Example 2 39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin

39-Desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin wassynthesised from 39-desmethoxyrapamycin according to the followingprocedure.

2.1 Synthesis of 39-desmethoxy-28-O-trimethylsilyl rapamycin

39-Desmethoxyrapamycin (170 mg, 0.17 mmol) and imidazole (51 mg, 0.75mmol) were dissolved in 5 mL ethyl acetate at 0° C. To this coldsolution chlorotrimethylsilane (77 mg, 0.09 mL, 0.71 mmol) was addeddrop wise over a period of 10 min. Stirring was continued for additional60 min to complete the formation of the 28,39-bis-O-trimethylsilylether. After that period 0.4 mL aqueous 0.5 N sulfuric acid was addedand the mixture was stirred for 2.5 h at 0° C. 20 mL ethyl acetate wasadded and the organic layer was washed with brine, saturated sodiumhydrogen carbonate solution and water. Drying over sodium sulfate andconcentration under reduced pressure yielded the 28-O-trimethylsilylether as a colourless solid which was used without further purificationfor the subsequent reaction.

¹H-NMR (400 MHz, CDCl₃), δ (ppm): 4.07 (d, 1H, J=6.5 Hz, C(28)-H), 0.00(s, 9H, 28-O-TMS). MS (ESI) m/z 978 [M+Na]⁺

2.2. Synthesis of 2,4,6-trichlorobenzoic2′,2′,5′-trimethyl-1′,3′-dioxane-5′ carboxylic anhydride

2,2-Dimethoxypropane (13.5 g, 130 mmol) and p-toluenesulfonic acidmonohydrate (100 mg, 0.53 mmol, 0.4 mol %) were added to a solution of2,2-bis(hydroxymethyl)propionic acid (13.5 g, 100 mmol) in acetone (100mL). The reaction mixture was stirred at room temperature for 2 h. Afterthat period moist sodium hydrogencarbonate was added and the mixture wasstirred for further 5 minutes. The supernatant was decanted off andconcentrated under reduced pressure. The resulting solid was treatedwith diethyl ether (3×50 mL) and the combined organic extracts wereconcentrated under reduced pressure to yield a white solid, 16.2 g (93%)

¹H-NMR (400 MHz, CDCl₃), δ (ppm): 4.19 (d, 1H, J=12.0 Hz) 3.68 (d, 1H,J=12.0 Hz) 1.45 (s, 1H) 1.41 (s, 1H) 1.20 (s, 1H).

This material was then converted into an activated mixed anhydride bythe method of U.S. Pat. No. 5,362,718. Thus, the acetonide (1.04 g, 5.98mmol) was dissolved in THF (20 mL) cooled to 0° C. and treated with thedropwise addition of triethylamine (0.83 mL, 5.98 mmol) and2,4,6-trichlorobenzoyl chloride (0.93 mL, 5.98 mmol). The reaction wasthen stirred at room temperature for 5 hours. The resulting precipitatewas filtered and washed with THF (10 mL). The combined filterate wasreduced in vacuo to a white amorphous solid which was used (as below)without further purification.

2.3. Synthesis of 39-desmethoxyrapamycin 28-O-trimethylsilyl ether,40-ester with 2,2,5-trimethyl[1.3-dioxane]-5-carboxylic acid

Crude 28-O-trimethylsilyl-39-desmethoxyrapamycin (200 mg, from 0.17 mmol39-desmethoxyrapamycin) from example 2.1 was dissolved in 2 mLdichloromethane. The solution was cooled to 0° C. and DMAP (102 mg, 0.84mmol) was added. Then, a solution of 2,4,6-trichlorobenzoic2′,2′,5′-trimethyl-1′,3′-dioxane-5′ carboxylic anhydride (159 mg, 0.42mmol) in 1 mL dichloromethane was added over a period of 10 min. Thereaction mixture was stirred at 0° C. for 5 h and the conversion wasmonitored by LC/MS. The reaction mixture was diluted with 7 mL ofdichloromethane and quenched by addition of 5 mL water. The organiclayer was separated and washed successively with 0.5 N sulfuric acid,sodium hydrogencarbonate solution and water. Drying over sodium sulfateand concentration under reduced pressure gave the title compound ascolourless foam, which was used immediately without furtherpurification.

MS (ESI) m/z 1111 [M−H]⁻

2.4. 39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin

Crude 39-desmethoxyrapamycin-28-O-trimethylsilyl ether 40-ester with2,2,5-trimethyl[1.3-dioxane]-5-carboxylic acid from example 2.3 wasdissolved in 2 mL acetone and 0.5 mL of 0.5 N sulfuric acid was added.The reaction mixture was stirred for 5 h at room temperature andsubsequently neutralised by the addition of 5 mL saturated sodiumhydrogencarbonate solution and 5 mL water. The aqueous mixture wasextracted with ethyl acetate and the combined organic extracts weredried over sodium sulphate. Concentration under reduced pressure gave acolourless solid which was purified by size exclusion chromatography onSephadex LH20 using chloroform/heptane/ethanol (v:v:v 10:10:1) aseluents.

¹H-NMR (500 MHz, CDCl₃), δ (ppm): 4.72 (m, 1H, C(40)-H), 3.87 (m, 2H),3.69 (m, 2H), 1.03 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃), δ (ppm): 175.52,74.04 (C(40)), 68.73 (2C), 48.90, 17.09. MS (ESI) m/z 1023 [M+Na]⁺

Example 3 39-desmethoxy-40-O-(2-hydroxy)ethyl rapamycin 3.1.2-(tert-butyldimethylsilyl)oxyethyl triflate

A solution of 2-(tert-butyldimethylsilyl)-ethylene glycol (125 mg, 0.71mmol) and 2,6-lutidene (0.08 mL, 0.69 mmol) in 6 ml dichloromethane wascooled to −78° C. Trifluoromethanesulfonic anhydride (0.11 mL, 0.65mmol) was added over a period of 5 min and stirring was continued foradditional 15 min at −78° C. to complete the formation of the triflate.The triflate was used in situ for the reaction as described in 3.2below.

3.2. 40-O-[2-(tert-butyldimethylsilyl)]ethyl-39-desmethoxyrapamycin

39-Desmethoxyrapamycin (300 mg, 0.34 mmol) and 2,6-di-tert-butylpyridine(1.5 mL, 6.68 mmol) were treated with2-(tert-butyldimethylsilyl)oxyethyl triflate (0.65 mmol in 6 mLdichloromethane) at room temperature. This solution was concentrated toa third of its original volume with a gentle stream of nitrogen and theresulting suspension was stirred for further 72 h at room temperature.After that period saturated sodium hydrogencarbonate solution (5 mL) andwater (5 mL) were added and the mixture was stirred for 30 min. Theorganic layer was separated and the aqueous phase was extracted withethyl acetate (2×5 mL). The combined organic extracts were dried oversodium sulfate and concentrated under reduced pressure to give acolourless oil. Purification by column chromatography on silica using agradient from hexane to hexane/acetone (v:v 1:1) gave the product as acolourless solid.

¹H-NMR (500 MHz, CDCl₃), δ (ppm): 4.16 (d, 1H, J=6.5 Hz, C(28)-H), 3.73(t, 2H, J=5.7 Hz), 3.52 (t, 2H, J=5.7 Hz), 0.89 (s, 9H), 0.06 (s, 6H);¹³C-NMR (125 MHz, CDCl₃), δ (ppm): 76.61 (C-40), 69.31 (CH₂), 63.03(CH₂), 25.92 (3C), 18.36, −5.23 (2C). MS (ESI) m/z 1065 [M+Na]⁺

3.3. 39-Desmethoxy-40-O-(2-hydroxy)ethyl rapamycin

A solution of 40-O-[2-(tert-butyldimethylsilyl)]ethyl-39-desmethoxyrapamycin (160 mg, 0.15 mmol) in 2 mL acetone was treated with 0.3 mL of0.5 N sulfuric acid at room temperature. The solution was allowed tostand at room temperature for 3 h and was subsequently quenched by theaddition of 5 mL saturated sodium hydrogencarbonate solution and 10 mLwater. The aqueous mixture was extracted with ethyl acetate (3×10 mL)and the combined organic extracts were dried over sodium sulfate.Concentration under reduced pressure gave a colourless solid which wasfurther purified by HPLC (water/acetonitrile v:v 20/80).

¹H-NMR (500 MHz, CDCl₃), δ (ppm): 4.16 (d, 1H, J=6 Hz), 3.70 (m, 2H),3.57 (m, 2H), 3.20 (m, 1H, C(40)-H); ¹³C-NMR (125 MHz, CDCl₃), δ (ppm):78.65 (C-40), 77.20 (C-28), 68.93 (CH₂O), 62.10 (CH₂O). MS (ESI) m/z 951[M+Na]⁺

Example 4 39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycinthrough lipase catalysed esterification of 39-desmethoxyrapamycin

A mixture of 39-desmethoxyrapamycin (720 mg, 0.82 mmol), vinyl2,2,5-trimethyl[1.3-dioxane]-5-carboxylate (244 mg, 1.22 mmol), lipasePS-C “Amano” II (720 mg) and molecular sieves 0.5 nm (250 mg) inanhydrous tert-Butyl methyl ether (3.5 mL) was heated to 43° C. under anatmosphere of argon. After 48 h LC/MS monitoring showed completeconversion of the starting material. THF (10 mL) was added and themixture was filtered through a pad of celite. The enzyme was washed withTHF (2×10 mL) and the combined organic extracts were concentrated underreduced pressure. The residue was dissolved in THF (50 mL) and H₂SO₄ (15mL, 0.5 N) was added. The solution was allowed to stand at roomtemperature for 5 h and the reaction was subsequently quenched by theaddition of NaHCO₃ (50 mL, 5%) and brine (50 mL). The aqueous mixturewas extracted with EtOAc (3×100 mL) and the combined organic extractswere dried over MgSO₄. Removal of solvents gave the product assemi-solid. Purification by flash chromatography (hexane/acetone 1:1)gave the product as a colourless solid.

The NMR data are identical with that of example 2.4 MS (ESI) m/z 1022[M+Na]⁺ Fragmentation of the sodium adduct gave ions at m/z 850, 728,693, 614, 560, 545, 441 and 431 in accordance with the fragmentationpattern shown in FIG. 3.

Example 5 39-Desmethoxy-40-O-[2-hydroxyethyl3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin

A mixture of 39-Desmethoxy-40-O-(2-hydroxy)ethyl rapamycin (40 mg, 0.04mmol), vinyl 2,2,5-trimethyl[1.3-dioxane]-5-carboxylate (25 mg, 0.13mmol), lipase PS-C “Amano” II (40 mg) and molecular sieve 0.5 nm (40 mg)in anhydrous tert-Butyl methyl ether (2 mL) was heated to 43° C. underan atmosphere of argon. After 72 h LC/MS monitoring showed completeconversion of the starting material. THF (10 mL) was added and themixture was filtered through a pad of celite. The enzyme was washed withTHF (2×10 mL) and the combined organic extracts were concentrated underreduced pressure. The residue was dissolved in acetone (7.5 mL) andH₂SO₄ (2.5 ml, 0.5 N) were added. The solution was allowed to stand atroom temperature for 2 h and the reaction subsequently quenched by theaddition of sat. NaHCO₃ (10 mL) and water (10 mL). The aqueous mixturewas extracted with EtOAc (3×10 ml) and the combined organic extractswere dried over MgSO₄. Removal of solvents gave the product as yellowishsolid. Purification by preparative HPLC on a Phenomenex 21.2×50 mm Luna5 μm C18 BDS column using a gradient from 70:30 MeCN/water to 100% MeCNover 15 min gave the product as a colourless solid.

MS (ESI) m/z 1067 [M+Na]⁺ Fragmentation of the sodium adduct gave ionsat m/z 894, 772, 738, 614, 604, 589, 475 and 441 in accordance with thefragmentation pattern shown in FIG. 4.

Example 627-O-desmethyl-39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin6.1 27-O-desmethyl-39-desmethoxyrapamycin, 40-ester with2,2,5-trimethyl[1.3-dioxane]-5-carboxylic acid

A mixture of 27-O-desmethyl-39-desmethoxy rapamycin (30 mg, 0.034 mmol),vinyl 2,2,5-trimethyl[1.3-dioxane]-5-carboxylate (34 mg, 0.17 mmol),lipase PS-C “Amano” II (30 mg) and molecular sieve 0.5 nm (30 mg) inanhydrous tert-Butyl methyl ether (2 mL) was heated to 43° C. under anatmosphere of argon for 72 h. THF (10 mL) was added and the mixture wasfiltered through a pad of celite. The enzyme was washed with THF (2×10mL) and the combined organic extracts were concentrated under reducedpressure to give a yellowish semi-solid. Purification by flashchromatography using hexane:acetone (v:v 2:1) gave the product as a paleyellow solid.

MS (ESI) m/z 1049 [M+Na]⁺

6.227-O-desmethyl-39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin

The material from 6.1 was dissolved in acetone (6 mL) and H₂SO₄ (2 mL,0.5 N) was added. The solution was allowed to stand at room temperaturefor 2 h and the reaction subsequently quenched by the addition of sat.NaHCO₃ (10 mL) and water (10 mL). The aqueous mixture was extracted withEtOAc (3×10 mL) and the combined organic extracts were dried over MgSO₄.Removal of solvents gave the product as yellowish solid. Purification bypreparative HPLC on a Phenomenex 21.2×50 mm Luna 5 μm C18 BDS columnusing a gradient from 70:30 MeCN/water to 100% MeCN over 15 min gave theproduct as a colourless solid.

MS (ESI) m/z 1009 [M+Na]⁺ Fragmentation of the sodium adduct gave ionsat m/z 836, 679, 600, 560, 531, 431, 427 in accordance with thefragmentation pattern shown in FIG. 5 ¹H NMR (500 MHz, CDCl₃) δ ppm 4.73(m, 1H, C(40)-H), 4.32 (d, J=4.5 Hz, 1H, C(27)-H), 4.19 (d, J=4.5 Hz,1H, C(28)-H), 3.89 (m, 2H), 3.70 (m, 2H) 1.03 (s, 3H).

Example 7 In vitro evaluation of anticancer activity of39-desmethoxy-40-O-(2-hydroxy)ethyl rapamycin and39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin

In vitro evaluation of 39-desmethoxy-40-O-(2-hydroxy)ethyl rapamycin and39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin foranticancer activity in a panel of 12 human tumour cell lines in amonolayer proliferation assay was carried out as described as Protocol 1in the general methods above using a modified propidium iodide assay.

The results are displayed in Table 3 below; each result represents themean of duplicate experiments. Table 4 shows the mean IC₅₀ and IC₇₀ forthe compounds across the cell lines tested, with rapamycin shown as areference.

TABLE 3 Cell Growth (Test/Control (%) at drug concentration)39-desmethoxy-40-O-(2- 39-desmethoxy-40-O-[2,2- Rapamycin hydroxy)ethylrapamycin bis(hydroxymethyl)propionyl]rapamycin Cell line 1 μM 10 μM 1μM 10 μM 1 μM 10 μM SF268 53.5 46 63 7 62.5 12 251L 75.5 40 90 31 85.513 H460 67 66 76 25 66 12 MCF7 68.5 26.5 77 10 67 9 MDA231 67 63.5 70.513.5 MDA468 56.5 32 66 9 394NL 45 44 55 6 46 13 OVCAR3 69 69.5 85 9 73.539 DU145 50.5 54 56 7 62.5 13.5 LNCAP 61 34 49 18 48.5 20.5 A498 58.548.5 66 19.5 1138L 42 21.5 59 4 50 7.5

TABLE 4 39-desmethoxy-40- 39-desmethoxy-40-O- O-(2-hydroxy)ethyl[2,2-bis(hydroxymeth- Rapamycin rapamycin yl)propionyl]rapamycin MeanIC₅₀ 3.5 2.2* 2.0 (microM) Mean IC₇₀ 9.1 5.513* 4.5 (microM) *mean wasbased on the 9 cell lines for which data was available

Example 8 In Vitro Binding Assays

FKBP12

FKBP12 reversibly unfolds in the chemical denaturant guandiniumhydrochloride (GdnHCl) and the unfolding can be monitored by the changein the intrinsic fluorescence of the protein (Main et al, 1998). Ligandswhich specifically bind and stabilise the native state of FKBP12 shiftthe denaturation curve such that the protein unfolds at higherconcentrations of chemical denaturant (Main et al, 1999). From thedifference in stability, the ligand-binding constant can be determinedusing equation 1.

$\begin{matrix}{{\Delta\; G_{app}} = {{\Delta\; G_{D - N}^{H_{2}O}} + {{RT}\;{\ln\left( {1 + \frac{\lbrack L\rbrack}{K_{d}}} \right)}}}} & (1)\end{matrix}$where ΔG_(app) is the apparent difference in free energy of unfoldingbetween free and ligand-bound forms, ΔG_(D-N) ^(H) ² ^(O) is the freeenergy of unfolding in water of free protein, [L] the concentration ofligand and K_(d) the dissociation constant for the protein-ligandcomplex (Meiering et al, 1992). The free energy of unfolding can berelated to the midpoint of the unfolding transition using the followingequation:ΔG_(D-N) ^(H) ² ^(O)=m_(D-N)[D]_(50%)  (2)where m_(D-N) is a constant for a given protein and given denaturant andwhich is proportional to the change in degree of exposure of residues onunfolding (Tanford 1968 and Tanford 1970), and [D]₅₀% is theconcentration of denaturant corresponding to the midpoint of unfolding.We defined ΔΔG_(D-N) ^(L), the difference in the stability of FKBP12with rapamycin and unknown ligand (at the same ligand concentration),as:ΔΔG_(D-N) ^(L)=<m_(D-N)>Δ[D]₅₀%  (3)where <m_(D-N)> is the average m-value of the unfolding transition andΔ[D]₅₀% the difference in midpoints for the rapamycin-FKBP12 unfoldingtransition and unknown-ligand-FKBP12 complex unfolding transition. Underconditions where [L]>K_(d), then, ΔΔG_(D-N), can be related to therelative K_(d)s of the two compounds through equation 4:

$\begin{matrix}{{{\Delta\Delta}\; G_{D - N}^{L}} = {{RT}\;\ln\frac{K_{d}^{X}}{K_{d}^{rap}}}} & (4)\end{matrix}$where K_(d) ^(rap) is the dissociation constant for rapamycin and K_(d)^(X) is the dissociation constant for unknown ligand X. Therefore,

$\begin{matrix}{K_{d}^{X} = {K_{d}^{rap}{\exp\left( \frac{< m_{D - N} > {\Delta\lbrack D\rbrack}_{50\%}}{RT} \right)}}} & (5)\end{matrix}$

Fitting each denaturation curve generates values for m_(D-N) and [D]₅₀%,which can be used to calculate an average m-value, <m_(D-N)>, andΔ[D]_(50%), and hence K_(d) ^(X). The literature value of K_(d) ^(rap)of 0.2 nM is used.

In some cases, due to the low solubility of the test compound, lowerconcentrations of the test compound were used than in the rapamycincontrol experiment. In these cases, the differences between the testcompound concentration and the rapamycin control concentration weretaken into account using equation 6 below:

$\begin{matrix}{K_{d}^{X} = {K_{d}^{rap}\frac{\left( {1 + \lbrack L\rbrack_{x}} \right)}{\left( {1 + \lbrack L\rbrack_{rap}} \right)}{\exp\left( \frac{< m_{D - N} > {\Delta\lbrack D\rbrack}_{50\%}}{RT} \right)}}} & (6)\end{matrix}$

TABLE 5 FKBP-12 in vitro binding assay results Ligand FKBP12 [L] μMK_(d) (nM) Rapamycin 10 0.2 39-desmethoxy-40-O-(2-hydroxy)ethylrapamycin 1.6 3.7 39-desmethoxy-40-O-[2,2- 6.67 1.1bis(hydroxymethyl)propionyl]rapamycinmTOR

Inhibition of mTOR was established indirectly via the measurement of thelevel of phosphorylation of the surrogate markers of the mTOR pathwayand p70S6 kinase and S6 (Brunn et al., 1997; Mothe-Satney et al., 2000;Tee and Proud, 2002; Huang and Houghton, 2002).

HEK293 cells were co-transfected with FLAG-tagged mTOR and myc-taggedRaptor, cultured for 24 h then serum starved overnight. Cells werestimulated with 100 nM insulin then harvested and lysed by 3 freeze/thawcycles. Lysates were pooled and equal amounts were immunoprecipitatedwith FLAG antibody for the mTOR/Raptor complex. Immunoprecipitates wereprocessed: samples treated with compound (0.00001 to 0.003 mM) werepre-incubated for 30 min at 30° C. with FKBP12/rapamycin,FKBP12/39-desmethoxyrapamycin derivative or vehicle (DMSO), non-treatedsamples were incubated in kinase buffer. Immunoprecipitates were thensubject to in vitro kinase assay in the presence of 3 mM ATP, 10 mM Mn²⁺and GST-4E-BP1 as substrate. Reactions were stopped with 4× samplebuffer then subject to 15% SDS-PAGE, wet transferred to PVDF membranethen probed for phospho-4E-BP1 (T37/46). Western blot bands werequantitated by image analysis using Image J(http://rsb.info.nih.gov/ij/). FIG. 6A shows dose-response curves forrapamycin (filled squares) and39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin (filledtriangles). FIG. 6B shows dose-response curves for rapamycin (filledsquares) and 39-desmethoxy-40-O-(2-hydroxy)ethyl rapamycin (filledtriangles).

Alternatively, HEK293 cells were seeded into 6 well plates andpre-incubated for 24 h and then serum starved overnight. Cells werepre-treated with vehicle or compound for 30 min at 30° C., thenstimulated with 100 nM insulin for 30 min at 30° C. and lysed by 3freeze/thaw cycles and assayed for protein concentration. Equal amountsof protein were loaded and separated on SDS-PAGE gels. The protein waswet transferred to PVDF membrane then probed for phospho-S6 (S235/36) orphospho-p70 S6K (T389). Western blot bands were quantitated by imageanalysis using Image J (http://rsb.info.nih.gov/ij/).

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1. A compound having the formula (I):

wherein: X represents bond or CH₂; R₁ represents a keto group or (H,H);R₂ represents OH or OMe; R₃ represents H, OH or OMe; R₄ and R₅ eachindependently represent H or OH; R₆ represents —R₇, —C(O)R₇,—(CH₂)₂—O—[CR₂₁R₂₂—O]_(a)—C(O)—R₂₃; —CR₂₁R₂₂—O—C(O)—R₂₃; —POR₁₉R₂₀,—PO(OR₁₉)(OR₂₀) or Y—R₁₅; R₇ represents—(CR₈R₉)_(m)(CR₁₀R₁₁)_(p)CR₁₂R₁₃R₁₄; R₈ and R₉ each independentlyrepresent C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl, any of whichgroups may optionally be substituted with —PO(OH)₂, —CF₂PO(OH)₂, —OH,—COOH or —NH₂; or R₈ and R₉ each independently represent H,trifluoromethyl or F; R₁₀, R₁₁, R₁₂, R₁₃ and R₁₄ each independentlyrepresent C1-C4 alkyl, C2-C4 alkenyl or C2-C4 alkynyl, any of whichgroups may optionally be substituted with —PO(OH)₂, —CF₂PO(OH)₂, —OH,—COOH or —NH₂; or R₁₀, R₁₁, R₁₂, R₁₃ and R₁₄ may be independentlyselected from H, —(CR₈R₉)_(q)NH₂, —(CR₈R₉)_(q)OH, CF₃, F, COOH; or R₁₀and R₁₁ or R₁₂ and R₁₃ or R₁₃ and R₁₄ may be taken together with thecarbon to which they are joined to form a C3-C6 cycloalkyl or a 3- to6-membered heteroalkyl ring that contains one or more heteroatomsselected from N, O and S and that is optionally, substituted with up to5 —(CR₈R₉)_(q)OH, —(CR₈R₉)_(q)NH₂ or COOH groups; Y=bond, —C(O)—O—;—(CH₂)₂—O—C(O)—O—; R₁₅ represents

R₁₆ are each independently H or OH; R₁₇ is independently selected fromH, OH and NH₂; R₁₈ is independently selected from H, —CH₃, —CH₂OH and—COOH; provided however that no more than 2 groups selected from R₁₆,R₁₇ and R₁₈ represent H or CH₃; R₁₉ and R₂₀ each independently representH or C1-C4 alkyl or R₁₉ and R₂₀ together represent ═CH₂; R₂₁ isindependently selected from H, CH₃; R₂₂ is independently selected fromH, —CH₃, —CH═CH₂, —CH₂Cl, —CHCl₂, —CCl₃, —CH(OH)Me, —CH₂OH, —CH₂CH₃,—CH(Cl)Me; R₂₃ is independently R₇, Y—R₁₅ or a 5- or 6-membered aryl orheteroaryl ring optionally substituted with between one and three groupsselected from OH, F, Cl, Br, NO₂ and NH₂; a represents 0 or 1; m, p andq each independently represent an integer between 0-4; provided howeverthat the R₇ moiety does not contain more than 12 carbon atoms and doescontain at least one functional group selected from —PO(OH)₂,—CF₂PO(OH)₂, —COOH, OH or NH₂; or a pharmaceutically acceptable saltthereof.
 2. A compound according to claim 1, wherein R₆ is selected fromthe group consisting of —R₇, —C(O)R₇, and—(CH₂)₂—O—[CR₂₁R₂₂—O]_(a)—C(O)—R₂₃.
 3. A compound according to claim 2,wherein R₂₃ is selected from R₇ and Y—R₁₅.
 4. A compound according toclaim 1, wherein R₇ satisfies at least one feature selected from thegroup consisting of: a) R₇ contains 7 or fewer carbon atoms; b) R₇contains 5 or fewer carbon atoms; c) R₇ contains two groups selectedfrom —PO(OH)₂, —CF₂PO(OH)₂, —OH, —COOH and —NH₂; and d) R₇ contains atleast one functional group selected from —COOH, OH and NH₂.
 5. Acompound according to claim 1, wherein p, m, or both represents 0 or 1.6. A compound according to claim 1, wherein q represents 0, 1 or
 2. 7. Acompound according to claim 1, wherein R₁₁, R₁₂, or both represents H.8. A compound according to claim 1, wherein R₁₃ represents H or OH.
 9. Acompound according to claim 1, where p represents 1, and R₁₀ representsMe, OH or CH₂OH; R₁₁ represents Me, H or CH₂OH; R₁₁ represents Me, H orCH₂OH; or both R₁₀ and R₁₁ represent Me, OH, or CH₂OH.
 10. A compoundaccording to claim 1, where m and p both represent 0; R₁₂ and R₁₃ bothrepresent H and R₁₄ represents —(CR₈R₉)_(q)—OH where q=0 or 1 and R₈ andR₉ both represent H.
 11. A compound according to claim 1, where prepresents 1 and m represents 0, R₁₀ and R₁₁ both represent H, R₁₂represents H, R₁₃ represents H, OH or NH₂ and R₁₄ represents—(CR₈R₉)_(q)—OH where q=0 or 1 and R₈ and R₉ both represent H.
 12. Acompound according to claim 1, wherein R₆ is C(O)R₇; wherein R₇ isselected from —CH₂OH, —C(CH₂)₂CH₂OH, —CH(OH)—CH₂OH, —CH(CH₂OH)—CH₂OH and—C(CH₂OH)₂—CH₃.
 13. A compound according to claim 1 which is selectedfrom the group consisting of39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin,39-desmethoxy-40-O-(2-hydroxy)ethyl rapamycin,39-desmethoxy-40-O-[2-hydroxyethyl3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate] rapamycin,27-O-desmethyl-39-desmethoxy-40-O-[2,2-bis(hydroxymethyl)propionyl]rapamycin or a pharmaceutically acceptable salt thereof.
 14. A compoundaccording to claim 1 where R₆ represents —POR₁₉R₂₀ or —PO(OR₁₉)(OR₂₀).15. A compound according to claim 14 where R₁₉ and R₂₀ both representCH₃ or both represent CH₂CH₃.
 16. A compound according to claim 1 whereR₆ represents Y—R₁₅.
 17. A compound according to claim 16 wherein R₁₅ isselected from

wherein R₁₆ and R₁₇ are OH and R₁₈ is CH₂OH; or R₁₆ is OH, R₁₇ is NH₂and R₁₈ is CH₂OH; or R₁₆ and R₁₇ are OH and R₁₈ is COOH; or R₁₆ is OH,R₁₇ is NH₂ and R₁₈ is H; or R₁₆ is OH.
 18. A pharmaceutical compositioncomprising a compound according to claim 1, at least onepharmaceutically acceptable diluents or carriers.
 19. A process forpreparation of a compound of formula (I) according to claim 1, whichcomprises: (a) reacting a compound of formula (II):

where R_(A) represents H or (CH₂)₂—OH or a protected derivative thereofwith a compound of formula (III): HO—R₆ (III); or an activatedderivative thereof wherein the group R₆ is as defined above forcompounds of formula (I); or a protected derivative thereof; or (b)converting a compound of formula (I) or a salt thereof to anothercompound of formula (I) or another pharmaceutically acceptable saltthereof; or (c) deprotecting a protected compound of formula (I).
 20. Acomposition or kit of parts comprising (i) a compound according to claim1 and (ii) one or more other therapeutically effective agent(s).
 21. Thecomposition or kit of parts of claim 20 wherein the one or more othertherapeutically effective agent(s) are selected from methotrexate,leukovorin, adriamycin, prenisone, bleomycin, cyclophosphamide,5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine,vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate,anastrozole, goserelin, anti-HER2 monoclonal antibody, capecitabine,raloxifene hydrochloride, EGFR inhibitors, VEGF inhibitors, proteasomeinhibitors, hsp90 inhibitors, azathioprine, corticosteroids,cyclophosphamide, cyclosporin A, FK506, Mycophenolate Mofetil, OKT-3,ATG, amphotericin B, flucytosine, echinocandins, griseofulvin, animidazole and a triazole antifungal agent.
 22. A compound according toclaim 16, wherein R₁₅ is

wherein R₁₆ is OH.